MEDICAL 


Property  of 


School  of  Nursing 
University  Hospital 


BACTERIOLOGY   FOR   NURSES 


THE  MACMILLAN  COMPANY 

NEW  YORK  •    BOSTON  •    CHICAGO  •    DALLAS 
ATLANTA  •    SAN   FRANCISCO 

MACMILLAN  &  CO.,  LIMITED 

LONDON   •    BOMBAY   •    CALCUTTA 
MELBOURNE 

THE  MACMILLAN  CO.  OF  CANADA,  LTD. 

TORONTO 


BACTERIOLOGY  FOR 

NURSES 


BY 


MARY   A.   SMEETON 

B.Sc.  (COLUMBIA  UNIVERSITY)  R.N. 

FORMERLY  SUPERINTENDENT  OF  NURSES,  PRESBYTERIAN  HOSPITAL 

ALLEGHENY;    ASSISTANT  BACTERIOLOGIST,  NEW  YORK  STATE 

HEALTH  DEPARTMENT;    INSTRUCTOR  IN  BACTERIOLOGY 

NEW  YORK  UNIVERSITY  AND  BELLEVUE  MEDICAL 

SCHOOL;    BACTERIOLOGIST  INTERNATIONAL 

HEALTH  BOARD,  FRANCE 


THE   MACMILLAN   COMPANY 
1922 


COPTEIGHT,    1920, 

BY  THE  MACMILLAN  COMPANY. 


Set  up  and  electrotyped.    Published  August,  1920. 


J.  8.  Gushing  Co.  —  Berwick  &  Smith  Co. 
Norwood,  Mass.,  U.S.A. 


563 

( 

D 


PREFACE 

WHILE  bacteriology  is  one  of  the  most  recent  subjects  introduced 
into  the  Training  School  Curriculum  it  is  by  no  means  of  least  im- 
portance ;  indeed,  so  intimately  is  it  related  to  the  other  subjects 
that  with  the  exception  of  anatomy  and  materia  medica  it  may 
be  regarded  as  necessary  to  a  right  understanding  of  them  all. 

The  science  of  bacteriology  has,  within  recent  years,  developed 
so  rapidly  that  it  is  impossible  to  more  than  mention  certain 
phases  of  the  subject  in  so  limited  a  space.  That  branch  which 
is  of  the  greatest  interest  to  the  student  nurse,  namely,  the  study 
of  pathogenic  microorganisms,  comprises  the  greater  part  of  the 
book ;  at  the  same  time  an  effort  has  been  made  to  point  out  that 
the  ability  to  produce  disease  is  limited  to  comparatively  few 
species  and  that  by  far  the  greatest  number  of  these  infinitesimal 
forms  of  life  perform  beneficent  tasks. 

An  attempt  has  been  made  to  present  the  subject  in  as  clear 
and  interesting  a  form  as  possible  in  order  to  enable  the  student 
to  realize  the  almost  incredible  force  of  the  microscopic  world, 
a  force  so  powerful  both  for  good  and  ill,  and  to  place  within  her 
reach  by  increased  knowledge  the  means  of  combating  the  baneful 
effects  of  those  forms  with  which  she  is  most  likely  to  have  to  deal. 

M.  A.  SMEETON. 


.MIS'.) 


CONTENTS 

PART  I 

AFTER  [PAGE 

I.    BACTERIA      r     .....        , .    . .    .  , 1 

II.    FACTORS   INFLUENCING   BACTERIAL  GROWTH.    DISINFECT- 
ANTS.   RESULT  OF  BACTERIAL  GROWTH  ....  13 

III.  STERILIZATION    OF    GLASSWARE.    PREPARATION    OF    CUL- 

TURE MEDIA 24 

IV.  MICROSCOPIC  EXAMINATION  AND  STAINING  OF  BACTERIA    .  38 
V.    CULTIVATION  AND  IDENTIFICATION  OF  BACTERIA         .        .  51 

VI.    BACTERIA  IN  NATURAL  PROCESSES  AND  INDUSTRIES   .        .  64 

VII.    BACTERIOLOGICAL  EXAMINATION  OF  WATER  AND  SEWAGE  .  74 

VIII.    MILK 83 

PART  II 

IX.    ABILITY  OF  BACTERIA  TO  PRODUCE  DISEASE       ...  94 

X.     BACTERIOLOGICAL  EXAMINATIONS 105 

XI.    BACTERIAL  TOXINS  AND  ANTITOXINS 112 

XII.     IMMUNITY 122 

XIII.  OPSONINS,  AGGLUTININS,  PRECIPITINS,  LYSIN      .        .        .  133 

XIV.  TYPES   OF  IMMUNITY.    PREPARATION   OF  VACCINE.     ANA- 

PHYLAXIS  .  143 


XV.    THE  PYOGENIC  Cocci 153 

XVI.    PNEUAIOCOCCUS,  MENINGOCOCCUS,  GONOCOCCUS  .        .        .  164 

XVII.    THE  DIPHTHERIA  BACILLUS 177 

XVIII.    THE  TUBERCLE  BACILLUS  AND  OTHER  ACID-FAST  ORGAN- 
ISMS             188 

XIX.     INTESTINAL  BACTERIA.    THE  COLON-TYPHOID  GROUP         .  200 
XX.     THE  COLON  TYPHOID  GROUP  (continued)    .         .         .         .208 

vii 


Vlll 


CONTENTS 


CHAPTER  PAGE 

XXI.    BACILLUS  ANTHRACIS.    BACILLUS  MALLEI.    BACILLUS  PYO- 

CYANEUS.    BACILLUS  PROTEUS         .        .        .        .        .221 
XXII.     (1)  HEMOGLOBINOPHILIC  GROUP.      (2)  HEMORRHAGIC  SEPTI- 

CEMIA  GROUP 232 

XXIII.  PATHOGENIC  ANAEROBIC  BACILLI 242 

XXIV.  THE  CHOLERA  SPIRILLUM  AND  ALLIED  ORGANISMS     .        .  253 
XXV.    PATHOGENIC  TRICHOMYCETES.    MOLDS.    YEASTS        .        .  266 

XXVI.    THE  PATHOGENIC  PROTOZOA.    AMEB^B.   FLAGELLATA  .  .    277 

XXVII.    SPOROZOA.    CILIATA      .        .        .        .        .        .        .  .289 

XXVIII.    DISEASES  CAUSED  BY  FILTRABLE  VIRUSES.    DISEASES  OF 

UNKNOWN  ETIOLOGY        .        .  300 


BACTERIOLOGY    FOR   NURSES 


CHAPTER  I 

BACTERIA 

CLASSIFICATION  —  STRUCTURE  —  COMPOSITION 

AMONGST  the  lowest  forms  of  living  things  organisms  exist  which 
are  too  small  to  be  seen  without  the  aid  of  a  powerful  micro- 
scope and  of  such  simple  structure  that  a  single  cell  suffices  for 
all  their  vital  activities.  Amongst  these  unicellular  organisms 
the  divergence  between  animal  and  plant  melts  away,  and  forms 
are  found  which  possess  minor  characteristics  of  both  groups. 
Prior  to  the  seventeenth  century  little  was  known  of  these 
minute  living  cells.  Conjectures  had  been  made  as  to  the  possi- 
bility of  their  existence  and  the  role  they  might  be  supposed  to 
play  in  various  natural  processes  and  in  disease,  but  they  were 
merely  conjectures  and  no  record  of  trustworthy  systematic  in- 
vestigations exists. 

Kircher  in  1659  observed  their  presence  in  putrid  meat  and 
milk ;  later,  in  1683,  Anton  van  Leeuwenhoeck,  a  Dutch  micros- 
copist,  recording  his  observations  upon  tartar  scraped  from  the 
teeth  and  mixed  with  water,  wrote,  "  With  the  greatest  astonish- 
ment I  saw  distributed  everywhere  through  the  material  I  was 
examining  '  animalcules '  of  the  most  microscopic  size  which 
moved  themselves  about  very  energetically."  Leeuwenhoeck 
supplemented  his  observations  with  drawings,  both  of  which  are 
remarkably  clear  and  accurate. 


2  BACTERIOLOGY  FOR  NURSES 

During  the  following  one  hundred  and  fifty  years  little  advance 
was  made.  Observers,  for  the  most  part,  were  content  with  simply 
seeing  these  minute  organisms  and  marveling  at  the  wonders  of 
nature. 

In  1762  Marcus  Antonius  von  Plenciz,  a  physician  of  Vienna, 
published  his  views  on  the  germ  theory  of  infectious  diseases. 
He  insisted  that  an  infectious  disease  had  as  its  cause  its  own 
specific  germ  and  that  infective  material  must  contain  the  living 
causal  agent  of  the  disease. 

A  decided  advance  was  made  by  Ehrenberg.  In  his  principal 
work  published  in  1838  upon  "  infusion  animals  "  he  described 
the  difference  between  the  larger  forms  and  conferred  upon  his 
"  animals "  some  of  the  names  still  current  in  bacteriological 
nomenclature. 

Very  soon  the  question  arose  as  to  the  origin  of  these  micro- 
organisms. Were  they  reproduced  from  similar  preexisting  forms 
(the  so-called  vitalistic  theory)  or  were  they  the  result  of  spon- 
taneous generation  due  to  changes  in  the  material  in  which  they 
were  found.  Liebig  and  his  supporters  held  the  view  that  fer- 
mentation and  putrefaction  were  simply  chemical  processes,  and 
that  all  albuminoid  bodies  would  if  left  to  themselves  disintegrate 
into  smaller  molecules.  The  force  of  Liebig' s  authority  over- 
shadowed for  some  time  the  vitalistic  theory  until  Pasteur  (1822- 
1895)  proved  that  albuminous  material  had  no  natural  tendency 
to  disintegrate,  and  that  putrefaction  and  decay  did  not  produce 
"  spontaneous  generation  of  life,"  but  on  the  contrary  were  manifes- 
tations of  the  presence  of  living  and  growing  organisms  engaged 
in  satisfying  their  need  of  food,  and  that  like  all  larger  animals 
and  plants  these  organisms  come  into  existence  only  by  means  of 
reproduction. 

As  a  result  of  the  researches  of  Pasteur  the  study  of  the  causal 
relation  of  bacteria  to  disease  was  taken  up  with  renewed  vigor. 
Investigations  into  the  cause  of  certain  infectious  diseases  in  plants 
and  insects  placed  the  doctrine  upon  a  firm  foundation ;  later 
it  was  demonstrated  that  microorganisms  were  responsible  for 
certain  infectious  diseases  in  man  and  animals  also.  To  Davaine, 


BACTERIA  3 

a  famous  French  physician,  belongs  the  honor  of  demonstrating 
the  latter  fact.  An  organism  was  found  in  the  blood  of  animals 
suffering  from  anthrax  by  Pollender  in  1849  and  by  Davaine  in 
1850.  It  was  the  latter,  however,  who  demonstrated  in  1863  by 
inoculation  experiments  that  the  specific  organism  was  the  cause 
of  the  anthrax. 

The  brilliant  researches  of  Pasteur  may  be  regarded  as  the 
foundation  of  the  Science  of  Bacteriology ;  later  investigators  have 
contributed  largely  to  placing  it  on  the  basis  of  an  independent 
position  in  natural  science.  A  great  impetus  was  given  to  the 
study  when  Robert  Koch  invented  a  solid-culture  medium  by  means 
of  which  the  descendants  of  a  single  cell  could  be  studied  alone. 
This  made  possible  the  knowledge  of  such  fundamental  principles 
as  the  mode  of  development,  physiological  requirements,  and 
capabilities  of  each  species,  a  knowledge  essential  not  only  to  a 
proper  understanding  of  bacteriology,  but  also  to  the  practical 
application  of  furthering  the  usefulness  of  such  microorganisms 
as  are  of  benefit  to  mankind  and  of  combating  those  which  by  their 
activities  produce  disease. 

Several  attempts  have  been  made  to  provide  a  satisfactory 
classification  of  these  microscopic  living  cells,  but  as  yet  no  one  has 
succeeded  in  presenting  a  really  adequate  one.  This  can  readily 
be  understood  when  one  realizes  the  minuteness  of  their  size  and 
the  consequent  difficulty  of  determining  their  relation  one  to  the 
other. 

In  addition  to  the  organisms  which  may  be  studied  by  means  of 
the  microscope  still  others  exist  which  are  so  small  as  to  be  in- 
visible with  any  magnification  which  we  now  possess.  That 
they  exist  is  certain  because  they  can  be  grown  in  mass  on 
culture  media  and  the  cultures  when  inoculated  into  susceptible 
animals  produce  the  characteristic  disease;  they  are  so  minute 
that  they  will  pass  through  the  finest  porcelain  filter.  The  group 
is  generally  spoken  of  as  Ultramicroscopic  or  Filtrable  viruses. 

The  following  broad  outline  (after  Park  and  Williams)  serves 
to  show  the  relationship  of  those  forms  that  are  of  special  interest 
in  that  they  are  able  to  produce  disease. 


BACTERIOLOGY   FOR   NURSES 


Classes  in  which 

Genera  in  which 

Kingdom          Subkingdom           pathogenic  species 

chief  pathogenic 

occur 

species  occur 

Cocci 

Micrococcus,         Diplo- 
coccus,       Streptococ- 
cus,   Staphylococcus, 

etc. 

Bacteria 

Bacilli 

Bacillus 

Plants      i 
(Fungi) 

(Schizomycetes) 

Spirilla 
Trichobacteria 

Spirillum      (Spirocheta, 
Treponema) 
Leptothrix,   Cladothrix, 
Nocardia,         Actino- 
myces 

Molds 
(Hyphomycetes) 

Mycomycetes 
Phycomycetes 
Unclassified  (Fungi 
Imperfect!) 

Aspergillus,  Penicillium 
Mucor 
Trichophyta,     Achoria, 
Microspora,  Sporotri- 

Yeasts  [  Oidia 

(Blastomycetes)  \  Saccharomycetes 


cha 
Oidium 
Saccharomyces 


Unclassified  Ultramicroscopic  Organisms 


Animals 
(Protozoa) 


Sarcodina 
Mastigophora 

Sporozoa 


Infusoria 


Rhizopodia 
Flagellata 

f  Telosporidia 
[  Neosporidia 
Ciliata 

BACTERIA 


Entameba 
Trypanosoma, 


Leish- 


Coccidium,       Sarcospo- 

ridium 
Nosema,  Babesia,  Plas- 

modium 
Balantidium 


Morphological  Relations.  —  Bacteria,  the  lowest  of  all  the 
microorganisms  known,  are  generally  classed  as  plants.  Their 
relationship,  however,  is  by  no  means  clearly  defined.  Like 
members  of  the  vegetable  kingdom  .certain  species  have  the 
ability  to  use  as  food  such  simple  elements  as  inorganic  carbon 
and  nitrogen;  on  the  other  hand  certain  species  show  a  resem- 
blance to  the  animal  kingdom  in  requiring  complex  organic  food. 
The  non-motility  of  some,  and  the  tendency  to  a  thread-like 
growth,  suggests  their  relationship  to  plant  life;  the  motility  of 
others  and  the  fact  that  none  possess  chlorophyl,  the  green  color- 
ing of  plants,  suggest  a  kinship  to  the  animal  kingdom.  It  is  best 


BACTERIA  5 

to  think  of  them  as  a  group  of  single-celled  organisms  probably 
representing  primitive  forms  that  existed  before  differentiation 
into  animal  and  vegetable  kingdoms  occurred. 

Classification.  —  Bacteria  may  be  divided  into  two  subgroups, 
a  lower  and  a  simpler  form  and  a  higher  and  more  developed  one. 
The  members  of  the  lower  form  are  minute  masses  of  protoplasm 
surrounded  by  an  envelope^  each  cell  a  living  unit  containing 
all  the  vital  capacities  of  an  independent  organism. 

Although  there  are  hundreds  of  different  species  there  are  only 
three  general  forms,  spheres  (cocci),  rods  (bacilli),  and  spirals 
(spirilla).  The  spheres  may  be  large  or  small,  and  may  group 
themselves  differently;  the  rods  may  be  long  or  short,  thick  or 
slender,  the  ends  may  be  rounded  or  sharply  rectangular ;  the  spirals 
may  be  flexible  or  stiff,  they  may  have  one,  two,  or  many  coils, 
but  still  spheres,  rods,  and  spirals  comprise  all  types.  So  far  as 
is  known,  it  is  never  possible  by  any  means  to  permanently  change 
the  form  of  the  members  of  one  group  to  that  of  another ;  that 
is,  under  suitable  conditions  cocci  always  produce  cocci,  bacilli 
always  produce  bacilli,  and  spirilla  always  produce  spirilla. 

The  higher  bacteria  (trichobacteria)  show  an  advance  on  the 
lower  forms  in  that  they  consist  of  united  segments,  branched  or 
unbranched,  which  are  surrounded  by  a  sheath  and  which  are 
more  or  less  interdependent. 

Transition  forms  no  doubt  exist  between  the  lower  and  higher 
forms,  and  for  this  reason  certain  organisms  are  difficult  to  classify. 
The  tubercle  bacillus,  for  example,  under  ordinary  conditions  is  a 
typical  rod  but  sometimes  it  produces  branching  filaments,  and 
for  that  reason  it  is  classed  by  some  writers  with  the  higher  bac- 
teria. 

THE  LOWER  BACTERIA 

Terminology.  —  The  terms  microbe,  microorganism,  and  germ 
are  frequently  used  to  designate  bacteria ;  they  may,  however,  be 
applied  to  any  form  of  microscopic  life. 

The  name  bacterium  is  given  to  any  single  member  of  the  group 
of  bacteria,  regardless  of  its  own  form. 


6  BACTERIOLOGY  FOR  NURSES 

Three  terms  are  in  use  to  designate  the  spirilla,  i.e.,  vibrio, 
spirillum,  and  spirochete.  According  to  Migula  the  names  are 
made  to  indicate  the  possession  and  arrangement  of  flagella. 
Fliige,  another  systematist,  applies  the  term  "  vibrio  "  to  all  forms 
that  are  slightly  curved,  and  "  spirillum  "  and  "  spirochete  "  to 
all  wavy  forms.  The  classification  of  this  group  is  at  present  an 
open  question. 

Size.  —  In  size  bacteria  vary  greatly.    The  cocci  range  from  l 

0.5/*  (  r  nnnn  inch)  to  2/*  (  inch)  in  diameter.     The  small- 

oOuuu 


est  bacillus  known,  the  influenza  bacillus,  has  an  average  size 
of  1/*X0.2/*.  The  largest  bacillus  recorded  (B.  Biitschlii)  is  50^ 
to  GO/*  long  and  4^  to  5/t  wide. 

Reproduction.  —  One  of  the  most  characteristic  features  of 
bacteria  is  their  method  of  reproduction.  They  multiply  by 
simple  division  or  fission  (hence  the  term  Schizomycetes  or  fission 
fungi).  This  method  of  multiplication  is  the  distinguishing 
feature  which  separates  bacteria  from  yeast,  the  latter  plants 
multiplying  by  a  process  known  as  budding.  When  a  bacterial 
cell  is  about  to  divide  a  constriction  appears  j^  thp-miflfjj*'  which 
gradually  becomes  more  pronounced  until  the  cell  is  completely 
divided  and  two  individuals  can  be  recognized.  These  may  be- 
come detached  at  once,  or,  owing  to  the  slimy  envelope  which  is 
more  or  less  developed  in  all  bacteria,  may  remain  attached. 

Certain  cocci  divide  as  described  into  two  individuals  which 
separate  at  once  (micrococci)  ;  others  dividing  in  one  plane  remain 
attached  in  pairs  (diplococci),  or  in  shorter  or  longer  chains 
(streptococci)  ;  others,  dividing  in  two  directions,  one  at  right 
angles  to  the  other,  form  groups  of  four  (tetrads)  ;  others  divide 
in  three  directions  and  form  packets  in  cubes  of  eight  (sarcinse)  ; 
others  again  divide  in  any  axis  and  form  irregular  grape-like 
bunches  (staphylococci)  . 

Division  among  the  bacilli  and  spirilla  always  takes  place  at 
right  angles  to  the  long  axis  of  the  cell.  The  cells  of  the  bacilli 

1  1/*  =  1  micron  or  micromillimetre  =  —  —  mm.,  about  'OKnnn  inch. 

1  (MX) 


BACTERIA  7 

for  the  most  part  separate  at  once ;  occasionally,  however,  they 
are  found  adhering  in  pairs  (diplobacilli)  or  chains  (streptobacilli). 
Certain  spirilla  show  a  tendency  to  remain  attached.  Thus  when 
seen  through  the  microscope  a  single  cell  of  one  of  the  shorter 
forms  may  have  the  appearance  of  a  comma,  a  pair  the  letter  S, 


FIG.  1.  —  Cocci,  Bacilli,  and  Spirilla. 

and  the  union  of  several  elements  may  appear  as  a  long  spiral 
form  (Fig.  1). 

Under  favorable  conditions  cell  division  takes  place  very  rapidly. 
A  cell  may  reach  maturity  and  divide  in  from  twenty  to  thirty 
minutes.  It  has  been  estimated  that  if  bacterial  multiplication 
continued  unchecked  for  two  days  and  the  division  of  each  cell 
took  place  only  once  an  hour  the  descendants  of  a  single  cell  would 
number  281,500,000.  It  has  been  further  calculated  that  these 
281,500,000  bacteria  would  form  a  solid  pint  and  would  weigh 
about  a  pound.  Such  multiplication,  however,  does  not  actually 
take  place.  Bacterial  growth  may  be  checked  by  various  factors, 
such,  for  example,  as  unsuitable  temperature,  lack  of  food  and 
moisture,  the  disintegration  of  food  substances  into  various  injuri- 
ous products  such  as  acids  and  alkalies,  the  excretion  of  the  bac- 
teria, or  the  competition  of  other  organisms. 

Involution    Forms.  —  When    the    bacterial    environment    has 
become  unfavorable  to  growth  the  bacteria  may  show  extremely 
irregular    structures  quite   different   to    the        ,-,          (==^> 
original  forms.     Long  thread-like  organisms       ft  A   O  ^^  s? 
with  irregular  thickenings  may  develop  which  ^    Q^ 

assume  a  dumb-bell  or  flask-like  shape  (Fig.      Fio-  2.— involution 
2).    These  are  termed  involution  or  degenerate 
forms.     That  they  really  represent  degenerate  changes  is  shown 
by  the  fact  that  when  transferred  to  favorable  conditions  growth 
slowly  takes  place  into  typical  forms  again.     It  sometimes  hap- 
pens, however,  that  these  involution  forms  lose  certain  properties 
which  are  never  regained. 


8  BACTERIOLOGY  FOR  NURSES 

Mutations.  —  Another  and  apparently  inexplicable  variation 
sometimes  appears  which  must  be  distinguished  from  the  above. 
Bacteria  may  lose  or  gain  certain  properties  and  the  fact  may  be 
explained  by  the  minute  divergence  of  successive  generations. 
Sudden  changes  or  mutations  occur,  however,  that  cannot  be  thus 
explained.1  An  instance  is  recorded  in  which  daughter  cells  sud- 
denly developed  the  power  to  ferment  saccharose  and  raffinose. 
During  four  years  of  successive  transplanting  the  parent  strain 
did  not  acquire  the  property  and  the  mutation  strain  did  not 
lose  it. 

Structure  of  Bacterial  Cell.  —  The  internal  structure  of  bacteria 
corresponds  in  simplicity  to  their  external  form.  When  examined 
under  the  microscope  in  a  living  condition  they  appear  as  minute 
colorless  refractile  bodies.  In  order  to  study  their  structure 
advantage  has  been  taken  of  their  affinity  for  the  various  dyes 
which  are  used  to  stain  animal  cells ;  in  this  way  several  interest- 
ing points  have  been  determined.  When  stained  the  cell  appears 
finely  granular  or  almost  homogeneous.  Many  theories  have 
been  advanced  as  to  the  nature  of  the  cell  substance  or  endoplasm. 
The  one  most  generally  accepted  is  that  the  cell  body  consists 
almost  entirely  of  nuclear  material  with  varying  amounts  of  cyto- 
plasm and  that  the  nuclear  material,  instead  of  being  gathered  in  a 
compact  mass  or  nucleus  as  in  animal  cells,  is  distributed  through- 
out the  cell  in  a  finely  divided  condition. 

Encircling  the  endoplasm  is  a  covering  of  cytoplasm  very  simi- 
lar in  composition.  The  name  ectoplasm  is  generally  considered 
more  appropriate  for  this  outer  layer  than  cell  membrane;  it  is 
from  this  outer  covering  that  the  flagella  or  organs  of  locomotion 
supposedly  originate. 

In  addition  to  the  endoplasm  and  its  covering  of  ectoplasm,  many 
bacteria,  and  perhaps  all,  are  provided  with  a  surrounding  capsule 
often  of  considerable  thickness.  Organisms  in  which  it  is  specially 
conspicuous  present  a  more  or  less  slimy  appearance  and  appear 
to  be  embedded  in  what  seems  to  be  a  mass  of  jelly.  Such  a  mass 
is  spoken  of  as  a  zooglcea  mass;  the  individuals  are  known  as 
1  Jordan,  Proc.  Nat.  Acad.  of  Science,  1915,  1,  p.  160. 


BACTERIA  9 

capsulated  bacteria.  The  capsule  is  most  easily  demonstrated 
in  preparations  made  directly  from  animal  tissues  or  fluids,  where, 
when  stained,  it  can  be  seen  surrounding  the  cell  like  a  halo. 

Metachromatic  Granules.  —  In  certain  bacteria  granules  have 
been  observed  which  show  a  greater  affinity  for  nuclear  dyes  than 
does  the  surrounding  protoplasm.  They  are  called  metachromatic 
granules  from  the  fact  that  by  appropriate  methods  they  will 
retain  one  stain  while  the  rest  of  the  bacterial  cell  can  be  made  to 
take  another.  In  young  diphtheria  bacilli  they  are  often  very 
conspicuous  and  serve  as  an  aid  in  diagnosis.  Their  nature  and 
significance  have  not  yet  been  determined. 

Other  granules  have  been  described  which  have  been  shown 
to  consist  of  starch  or  fat  or  of  other  substances ;  they  probably 
represent  material  in  process  of  transformation  into  cell  nutrition. 
In  certain  bacteria  which  find  their  food  supply  in  decaying  organic 
material  granules  of  sulfur  have  been  demonstrated,  in  others 
iron  granules. 

Motility.  —  Many  species  of  bacteria  are  capable  of  independent 
movement.  When  seen  in  a  fluid  preparation  through  the  micro- 
scope their  movements  may  appear  of  a  darting  or  rolling  nature 
or  they  may  be  very  sluggish  and  scarcely  perceptible.  Bacterial 
motility  is  always  a  real  progressive  motion  and  not  merely  the 
oscillating  vibration  exhibited  by  all  finely  divided  particles  sus- 
pended in  suitable  fluid.  The  latter  so-called  "  Brownian  move- 
ment "  is  a  purely  physical  phenomenon  which  may  be  shown  by 
dead  bacteria  and  inorganic  substances. 

The  speed  with  which  certain  bacteria  move  has  been  estimated ; 
the  cholera  spirillum,  for  example,  may  travel  for  a  short  distance 
at  the  rate  of  18  cm.,  or  about  7  inches,  per  hour.  While  this 
does  not  seem  very  great  it  is  considerable  when  one  considers 
the  minute  size  of  the  organism.  Most  of  the  actively  motile 
bacteria  are  bacilli  or  spirilla. 

The  motility  of  bacteria  depends  upon  their  possession  of  thin 
hair-like  appendages  or  flagella  which  are  so  extremely  fine  that 
special  staining  methods  are  necessary  to  demonstrate  them. 
By  means  of  their  power  of  contractility  they  keep  up  a  lashing 


10 


BACTERIOLOGY   FOR   NURSES 


to  and  fro  movement  which  serves  to  propel  the  bacterium  through 
the  liquid  in  which  it  is  growing  (Fig.  3). 


FIG.  3.  —  Arrangement  of  Flagella. 

The  arrangement  of  the  flagella  varies  in  the  different  species  of 
bacteria.  Following  is  a  classification  according  to  their  number 
and  distribution : 


SPECIES 

DESCRIPTION 

EXAMPLE 

Monotricha 
Amphitricha 
Lophotricha 
Peritricha 

Atricha 

A  single  flagellum  at  one  pole 
A  flagellum  at  each  pole 
A  tuft  of  flagella  at  one  pole 
Flagella  projecting  from  all  parts  of 
the  surface 
No  flagella 

Cholera  spirillum 
Many  spirilla 
Spirillum  undula 
Typhoid  bacillus 

All  non-motile  bacteria 

The  degree  of  motility  depends  upon  the  species,  the  age  of 
growth,  heat,  light,  the  presence  of  chemicals,  etc.  The  property 
by  means  of  which  bacteria  are  aware  of  the  various  forces  which 
influence  them  is  known  as  taxis.  When  they  are  attracted  the 
phenomenon  is  spoken  of  as  positive  taxis,  when  they  are  repelled 
as  negative  taxis. 

Spore  Formation.  —  Under  certain  circumstances  some  species 
of  bacteria  produce  changes  in  their  protoplasm  which  result 
in  the  formation  of  bodies  known  as  endospores,  and  to  these 
new  bodies  all  the  vital  powers  of  the  original  cell  are  transferred. 
Its  commencement  in  a  bacterium  is  indicated  by  the  endoplasm 
becoming  turbid  and  the  appearance  of  minute  refractile  granules 
which  do  not  readily  take  the  ordinary  stains.  By  degrees  the 
granules  become  larger  and  finally  coalesce  into  a  spherical  or 
oval  body,  always  shorter  but  often  broader  than  the  original 
bacterium.  Surrounding  the  endospore  is  a  dense  protective 
envelope  which  is  supposed  to  give  to  the  spore  its  characteristic 


BACTERIA  11 

property,  namely,  great  resistance  to  harmful  influences  such  as 
heat,  chemicals,  etc. 

The  spore  may  be  formed  in  any  part  of  the  bacterium  but  its 
position  is  generally  constant  in  the  same  species.  It  may  lie 
within  the  center  of  the  cell  without  changing  the  contour  of  the 
latter,  or  it  may  distend  the  central  part  of  the  cell,  giving  it  a 
spindle-like  shape,  or  it  may  be  formed  at  one  end,  giving  the 
bacterium  the  appearance  of  a  drumstick. 

When  conditions  again  become  favorable  for  growth,  the  or- 
ganism assumes  its  original  form.  The  spore  absorbs  moisture, 
becomes  swollen,  and  loses  its  slimy  refractile  appearance ;  later  a 
little  bulging  is  seen  on  one  side  of  the  cell  if  the  spore  is  central 
or  at  the  extremity  if  the  spore  is  polar.  This  protrusion  continues 
until  finally  the  spore  envelope  bursts  and  a  rod  of  soft  protoplasm 


(i) 

FIG.  4. —  (a)  Position  of  Spores.     (6)  Germination  of  Spores. 

emerges  which  then  commences  to  function  in  the  ordinary  manner 
of  its  species  (Fig.  4). 

Spore  formation  must  not  be  regarded  as  a  method  of  reproduc- 
tion ;  it  is  a  resting  stage  which  should  be  contrasted  with  the 
vegetative  stage  when  active  multiplication  takes  place.  It  occurs 
most  frequently  in  bacilli,  less  often  in  spirilla  and  very  rarely 
in  cocci.  Fortunately  there  are  very  few  spore-bearing  organisms 
pathogenic  for  man,  a  fact  which  greatly  simplifies  disinfection 
and  the  treatment  of  infectious  diseases. 

Two  views  are  advanced  regarding  the  significance  of  spore 
formation  in  bacteria.  According  to  one  view  it  is  considered  as 
a  period  of  rejuvenescence  and  that  an  alternation  between  the 
vegetative  and  spore  stage  is  necessary  in  order  that  the  species 
may  maintain  its  highest  vitality.  In  support  of  this  view  there 
exists  the  fact  that  in  some  cases  sporulation  will  cease  at  a  temper- 
ature above  or  below  the  optimum,  while  vegetative  growth  will 
have  a  much  longer  range.  The  anthrax  bacillus  if  kept  at  a 


12  BACTERIOLOGY  FOR  NURSES 

temperature  above  the  limit  at  which  it  grows  best  not  only  ceases 
to  form  spores  but  it  loses  its  power  of  sporulation. 

The  second  view  is  that  a  bacterium  only  forms  spores  when 
conditions  are  unfavorable  to  its  life  and  growth ;  that  it  is  essen- 
tially a  process  whereby  a  species  may  be  preserved  in  a  hostile 
environment  until  its  surroundings  again  become  favorable.  The 
lack  of  food,  the  presence  of  substances  excreted  by  the  bacteria 
themselves,  and  the  products  formed  by  the  disintegration  of  the 
food  material  in  which  they  are  growing  play  an  important  part 
in  making  unsuitable  surroundings.  Species  which  form  spores 
under  these  conditions  will  always  change  into  vegetative  forms 
when  placed  in  a  fresh  food  supply. 

The  tests  usually  made  to  determine  whether  or  not  spore 
formation  has  taken  place  are :  (1)  Subjection  of  the  culture  to  a 
temperature  high  enough  to  kill  vegetative  forms  without  injuring 
spores,  (2)  Microscopic  examination  for  the  presence  of  a  refractile 
body  within  the  bacterium,  which  cannot  be  stained  by  ordinary 
dyes  but  which  can  be  colored  by  the  special  methods  devised  for 
the  staining  of  spores  (page  30). 

Chemical  Composition.  —  The  chemical  composition  of  bacteria 
varies  somewhat  according  to  the  nature  of  the  species  and  the 
material  upon  which  they  are  growing.  Ordinarily  the  bodies 
of  bacteria  contain  from  80  to  88  per  cent  of  water.  Substances 
of  a  protein  nature  similar  to  the  albumins  and  globulins  found  in 
animal  and  plant  tissues  are  present  which  probably  represent 
the  vitality  of  the  cell.  The  presence  of  fat  has  been  demonstrated ; 
also  starch-like  granules  staining  blue  with  iodine  have  been 
observed.  Sulphur,  iron,  calcium,  potassium,  chlorin,  magnesium, 
etc.,  may  also,  in  small  quantities,  form  part  of  the  bacterial 
protoplasm. 


CHAPTER  II 

FACTORS  INFLUENCING  BACTERIAL  GROWTH. 
DISINFECTANTS.     RESULTS  OF  BACTERIAL  GROWTH 

Habitat.  —  Bacteria  of  one  species  or  another  exist  almost  every- 
where. Forty  different  forms  have  been  described  as  common 
in  soil.  They  are  present  in  the  air  in  large  numbers  in  populated 
areas,  especially  near  the  surface  of  the  ground.  Particles  of  dust 
may  be  laden  with  them ;  anything  that  tends  to  set  the  dust 
in  motion  considerably  increases  the  number  of  bacteria  in  the 
air,  hence  the  necessity  of  sprinkling  floors  before  sweeping  and 
"  moist  "  dusting  whenever  possible.  They  are  present  in  the 
mouth,  stomach,  and  intestines  of  animals  and  human  beings; 
on  the  surface  of  the  skin,  under  the  finger  nails,  and  on  the  hairs. 
They  are  present  in  rivers,  and  in  the  ocean.  They  are  especially 
abundant  in  all  forms  of  decaying  matter.  Everywhere,  then, 
in  nature  there  exists  this  tremendous  force  with  its  wonderful 
power  of  multiplication,  a  power  which  nevertheless  is  held  in 
control. 

FACTORS  INFLUENCING  BACTERIAL   GROWTH 

Food.  —  Perhaps  the  universality  of  bacteria  is  explained  by 
the  fact  that  they  can  utilize  the  most  diverse  substances  as  food, 
substances  varying  from  the  simplest  to  the  most  complex  nitrog- 
enous compounds.  The  presence  of  nitrogen,  however,  in  some 
form  is  indispensable. 

A  simple  classification  of  bacteria  may  be  made  on  the  basis 
of  their  food  requirements.  Those  which  supply  their  nutritional 
needs  while  engaged  in  disintegrating  the  lifeless  remains  of  plants 
or  animals  are  known  as  strict  saprophytes.  On  the  other  hand, 

13 


14  BACTERIOLOGY   FOR   NURSES 

those  species  which  can  grow  only  within  or  upon  a  living  host 
(plant  or  animal)  are  known  as  strict  parasites.  A  hard  and  fast 
line  cannot  be  drawn  between  these  two  groups  because  there 
exist  certain  organisms  which  are  ordinarily  saprophytes  but 
which  may  grow  in  living  tissues  and  cause  disease.  Such  forms 
are  spoken  of  as  facultative  parasites.  Other  species  exist  which 
grow  best  as  parasites  in  living  tissue  but  which  can  be  cultivated 
on  non-living  material.  The  pathogenic  organisms  which  can  be 
grown  on  culture  media  belong  to  this  class.  They  are  known  as 
facultative  saprophytes. 

Moisture.  —  Water  is  essential  for  bacterial  growth.  The  dif- 
ferent species  vary  in  the  degree  of  their  need.  The  cholera  spiril- 
lum, if  deprived  of  moisture,  will  die  in  from  two  to  three  hours; 
the  bacillus  of  diphtheria  may  live  under  the  same  conditions  several 
days.  Spores  are  much  more  resistant  to  drying  than  vegetative 
forms ;  they  will  germinate  after  remaining  in  a  dry  condition  for 
years.  It  often  happens,  however,  that  organisms  exposed  to  such 
harmful  influences,  even  though  they  survive,  lose  some  of  their 
original  properties. 

Osmosis.  —  A  certain  degree  of  dilution  is  necessary  for  food 
substances  in  solution.  When  bacteria  suddenly  find  themselves 
in  a  concentrated  fluid  they  cannot  readily  adjust  themselves, 
and  if  the  difference  is  too  sudden  or  too  great  death  may  speedily 
result.  An  illustration  of  this  is  the  keeping  qualities  of  a  thick 
syrup  as  compared  with  the  rapid  fermentation  of  a  dilute  sugar 
solution.  The  best  development  of  an  organism  takes  place  when 
the  osmotic  pressure  is  the  same  in  the  surrounding  fluid  as  that 
within  the  cell  itself.  If  the  fluid  is  too  concentrated  water  is 
drawn  from  the  bacterial  cell  and  the  protoplasm  shrinks  from  its 
outer  covering ;  the  condition  is  spoken  of  as  "  plasmolysis." 
If  on  the  other  hand  the  new  fluid  has  a  lower  pressure  the  cell 
absorbs  more  water  and  may  burst.  This  latter  condition  is 
termed  "  plasmoptysis." 

Oxygen.  —  The  free  oxygen  of  the  air  is  absolutely  necessary 
for  the  growth  of  the  majority  of  organisms ;  there  is,  however, 
a  small  group  which  cannot  live  when  it  is  present.  Pasteur 


FACTORS   INFLUENCING   BACTERIAL   GROWTH    15 

was  the  first  to  note  this  extraordinary  fact  and  he  suggested  the 
grouping  of  bacteria  into  two  divisions,  viz. :  aerobes  which  re- 
quire the  presence  of  free  oxygen,  and  anaerobes,  which  require  the 
exclusion  of  free  oxygen.  Midway  between  the  obligatory  aerobes 
and  the  obligatory  anaerobes,  however,  there  are  many  organisms 
which  do  not  belong  strictly  to  one  group  or  the  other.  Faculta- 
tive anaerobes  grow  best  in  the  presence  of  oxygen  but  their  growth 
is  not  checked  when  the  supply  is  limited.  Facultative  aerobes 
on  the  other  hand  are  anaerobes  that  can  tolerate  a  certain  amount 
of  free  oxygen.  Anaerobes  are  not  affected  by  the  presence  of 
hydrogen  or  nitrogen. 

Light.  —  The  effect  of  light  upon  bacteria  is  very  marked. 
Bright  daylight  may  inhibit  their  growth,  and  many  species  can- 
not live  when  exposed  to  the  full  action  of  the  sun's  rays.  A 
longer  exposure  is  necessary  when  they  are  moist  than  when 
they  are  dry.  Typhoid  bacilli  are  killed  in  about  one  and  a 
half  hours.  The  bactericidal  effect  of  light  is  due  mainly  to  the 
green,  violet,  and  ultraviolet  rays.  An  interesting  experiment 
illustrating  the  effect  of  light  upon  bacteria  may  be  carried  out  by 
pouring  inoculated  media  into  a  Petri  dish,  the  cover  of  which  has 
been  partly  shaded  by  pasting  on  a  strip  of  black  paper.  The 
plate  after  being  exposed  to  direct  sunlight  will  show  colonies  only 
in  the  shaded  portion.  A  series  of  plates  may  be  prepared  and 
exposed  varying  lengths  of  time,  half  an  hour,  one  hour,  etc. 
The  plates  should  be  kept  two  or  three  days  at  20°  C.  to  30°  C. 
to  allow  the  colonies  in  the  shaded  portion  to  develop. 

Electricity.  —  The  effects  of  electricity  upon  bacteria  have  not 
been  thoroughly  studied.  A  powerful  electric  light  is  supposed 
to  be  as  fatal  as  sunlight. 

Radium  and  Rontgen  rays  have  not  been  shown  to  have  more 
than  a  slight  inhibitory  action. 

Temperature.  —  The  range  of  temperature  within  which  bac- 
terial growth  of  one  species  or  another  may  occur  lies  between  0°  C. 
and  72°  C.  For  every  species  there  is  an  optimum  temperature 
or  a  temperature  at  which  its  growth  is  most  luxuriant.  Each 
species  too  has  its  maximum  temperature  above  which  growth 


16  BACTERIOLOGY  FOR  NURSES 

will  not  take  place  and  its  minimum  temperature  below  which  it 
is  inactive.  Death  does  not  necessarily  occur  at  these  limits 
but  reproduction  does  not  take  place.  The  maximum  and  mini- 
mum for  each  species  has  a  range  of  from  twenty  to  thirty  degrees 
and  its  optimum  does  not  extend  ordinarily  more  than  five  degrees. 
The  maximum  for  some  may  be  below  the  minimum  for  others, 
for  example,  for  B.  phosphorescens  the  minimum  temperature  at 
which  growth  occurs  is  0°  C.,  the  optimum  20°  C.,  and  the  maxi- 
mum 37°  C.,  while  for  B.  thermophilus,  an  organism  found  in  fer- 
menting manure,  the  minimum  temperature  is  40°  C.,  the  opti- 
mum about  66°  C.,  and  the  maximum  72°  C.  Generally  speaking, 
the  optimum  temperature  for  bacteria  is  the  ordinary  temperature 
of  their  natural  habitat.  The  most  favorable  temperature  then 
for  pathogenic  organisms  is  that  of  the  human  body.  If  grown  on 
culture  media  at  a  higher  temperature  they  may  lose  their  viru- 
lence. 

The  vegetative  forms  of  most  bacteria  are  killed  by  half  an  hour's 
exposure  in  the  presence  of  moisture  to  a  temperature  of  from 
55°  C.  to  58°  C.  or  by  10  minutes'  exposure  to  a  temperature  of 
60°  C.  to  80°  C.  There  are  no  non-spore-bearing  forms,  except 
a  few  cocci,  that  can  live  in  boiling  water  even  for  a  few  minutes. 
Most  of  the  pathogenic  bacteria,  including  the  cholera  spirillum, 
the  typhoid  bacillus,  and  the  tubercle  bacillus,  are  destroyed  in 
ten  minutes  when  exposed  to  moist  heat  at  60°  C.  Thus  milk 
properly  pasteurized  or  water  brought  to  the  boiling  point  are 
rendered  harmless  so  far  as  these  germs  are  concerned. 

Dry  heat  is  much  less  effective  than  moist ;  many  pathogenic 
organisms  can  withstand  in  the  absence  of  moisture  a  temperature 
of  100°  C.  for  half  an  hour.  Spores  are  especially  resistant  to  both 
dry  and  moist  heat.  Practically  all  forms,  however,  are  killed  by 
exposure  to  dry  heat  for  one  hour  at  150°  C.  or  to  steam  under 
pressure  in  an  autoclave  for  15  minutes  at  125°  C. 

Bacteria  are  affected  by  low  temperatures  much  less  than  by 
high.  Many  have  been  subjected  to  a  temperature  of  liquid  air 
(about  -190°  C.)  without  being  destroyed.1  In  a  culture  of 
1  Park  and  Williams,  "  Pathogenic  Microorganisms,"  p.  56. 


FACTORS   INFLUENCING   BACTERIAL  GROWTH     17 

typhoid  bacilli  exposed  to  -175°  C.  for  thirty  minutes  10  per  cent 
remained  alive. 

Antibiosis  and  Symbiosis.  —  In  nature  many  species  of  bacteria 
exist  side  by  side ;  pure  cultures  are  seldom  found  outside  of  the 
laboratory.  In  some  instances  the  products  of  one  species  are 
antagonistic  to  the  well-being  of  another  and  the  weaker  is  able 
to  multiply  very  slowly  or  not  at  all.  Saprophytic  forms  soon 
overpower  the  comparatively  less  resistant  disease-producing  forms. 
Thus  infected  carcasses  eventually  become  purified  by  the  same 
processes  that  destroyed  them. 

Sometimes,  on  the  contrary,  there  is  a  certain  amount  of 
cooperation  between  two  species  and  the  presence  of  one  induces 
the  more  luxuriant  growth  of  the  other.  This  latter  condition  is 
spoken  of  as  symbiosis.  Certain  anaerobes  will  grow  when  air 
is  admitted  into  the  culture  tube  if  cultivated  with  an  ae'robe; 
it  is  assumed  that  the  ae'robe  deprives  the  air  of  its  oxygen  con- 
tent and  thus  renders  conditions  suitable  for  anaerobic  growth. 

Effects  of  Chemicals.  —  Chemical  substances  vary  in  their 
bactericidal  powers  as  do  the  various  bacteria  in  their  degree  of 
resistance.  Just  what  their  action  really  is,  in  many  instances, 
is  not  known.  In  the  case  of  bichloride  of  mercury  or  formaldehyde 
there  appears  to  be  a  chemical  union- between  the  disinfectant  and 
the  cell  protoplasm.  Vegetative  forms  are  affected  more  quickly 
than  spores. 

The  following  terms  are  frequently  used  to  express  the  inimical 
effect  of  chemicals  or  physical  forces  upon  bacteria : 

Attenuation.  —  Function  diminished  but  not  impaired. 

Antiseptic  Action.  —  Growth  arrested  but  capable  of  recom- 
mencement as  soon  as  surroundings  become  suitable. 

Disinfection.  —  Destruction  of  all  disease-producing  forms  and 
their  products. 

Sterilization.  —  Destruction  of  all  forms,  pathogenic  and  non- 
pathogenic. 

Disinfectants  which  are  very  effective  under  certain  circum- 
stances may  become  almost  inert  under  others.  Milk  of  lime  is 
of  use  only  while  it  remains  milk  of  lime ;  when  the  carbon  dioxide 


18  BACTERIOLOGY  FOR  NURSES 

of  the  air  has  converted  it  into  carbonate  of  lime  it  is  practically 
harmless.  Bichloride  of  mercury  is  a  powerful  disinfectant  under 
some  circumstances  but  when  placed  in  contact  with  organic  ma- 
terial it  forms  an  albuminate  which  renders  it  much  less  effective. 
On  this  account  it  is  not  well  suited  to  the  disinfection  of  sputum 
and  feces. 

In  applying  a  disinfectant  whose  strength  is  known  it  should 
always  be  remembered  that  it  must  be  present  throughout  the 
entire  mass  in  the  proportion  required.  Thus  if  a  disinfectant  is 
active  in  a  10  per  cent  solution  it  cannot  be  used  in  that  strength 
to  disinfect  an  equal  volume  of  infected  material  —  the  mixture 
would  contain  only  5  per  cent  of  the  bactericidal  substance.  An 
equal  volume  of  a  20  per  cent  solution  would  be  required  to  give 
10  per  cent  of  the  disinfectant  in  the  resulting  mixture. 

Methods  for  the  standardization  of  disinfectants  have  been 
devised  whereby  their  relative  value  may  be  determined.  Car- 
bolic acid  is  used  as  the  standard  and  the  comparative  strength 
of  other  substances  is  stated  in  terms  of  their  coefficiency. 

Even  after  the  relative  strength  of  a  bactericidal  substance  is 
known  it  should  be  remembered  that  the  reaction  of  the  solution 
and  the  material  to  be  disinfected  must  be  considered.  Thus  if 
an  alkali  such  as  lime  is  used  to  disinfect  an  acid  substance,  suffi- 
cient lime  must  be  added  first  to  neutralize  the  acid  and  then  the 
additional  amount  required  for  disinfection  must  be  added. 

A  great  number  of  more  or  less  effective  disinfectants  have  been 
put  upon  the  market,  the  most  costly  of  which  are  by  no  means 
the  most  reliable.  Such  well-known  chemicals  as  carbolic  acid, 
bichlorid  of  mercury,  lime,  coal  tar,  creosotes,  formalin,  etc.,  give 
a  wide  range  of  choice  and  in  addition  their  advantages  and  limi- 
tations are  well  established. 

DISINFECTANTS 

Carbolic  Acid.— A  solution  of  1  part  to  1000  inhibits  the  growth 
of  bacteria,  1  part  to  100  kills  vegetative  forms  in  from  five  to 
thirty  minutes,  and  1  part  to  20  kills  most  spores  within  a  few  hours 
and  all  within  a  period  of  from  one  to  four  weeks.  Carbolic  acid 


DISINFECTANTS  19 

is  perhaps  one  of  the  most  generally  used  disinfectants  because 
it  is  so  little  affected  by  albuminous  substances,  it  is  not  readily 
decomposed,  and  does  not  harm  fabrics,  metals,  or  wood. 

Alcohol.  —  Ten  per  cent  solution  inhibits  bacterial  growth ; 
absolute  alcohol  kills  vegetative  forms  within  twenty-four  hours. 

Formalin  is  a  40  per  cent  solution  of  formaldehyde  gas;  it  is 
supposed  to  have  about  one  half  the  germicidal  power  of  carbolic 
acid ;  a  2  per  cent  solution  of  formalin  will  kill  vegetative  forms  in 
from  five  to  thirty  minutes. 

Formaldehyde  is  probably  of  greatest  service  in  its  gaseous 
form  as  a  disinfectant  of  buildings  and  furniture.  It  does  not 
harm  delicate  fabrics;  wood,  copper,  brass,  and  silver  are  not 
affected  by  it.  Under  ordinary  circumstances  its  powers  of  pene- 
tration are  not  great  and  it  can  only  be  depended  on  for  surface 
disinfection.  Vegetative  forms  of  bacteria  exposed  directly  to  the 
action  of  concentrated  formaldehyde  gas  are  killed  at  once.  It 
is  advisable,  however,  in  disinfecting  a  room  to  allow  several  hours' 
exposure  in  order  that  the  gas  may  reach  all  corners.  It  has  very 
little  effect  upon  animals  or  insects. 

lodoform  as  such  has  little  effect  upon  bacteria.  When  in  the 
presence  of  pus  it  is  broken  down  into  iodine  compounds  which 
act  partly  by  rendering  inert  the  poisons  produced  by  bacteria 
and  partly  by  destroying  the  bacteria  themselves. 

Chloroform  will  destroy  bacteria  in  the  vegetative  form  in  1 
per  cent  solution.  It  is  not  known  to  have  any  effect  upon  spores. 

Lysol  is  a  coal-tar  product  containing  about  50  per  cent  cresols. 
It  is  generally  used  in  1  per  cent  solution;  it  has  about  double 
the  strength  of  carbolic  acid. 

Bichloride  of  Mercury,  commonly  known  as  corrosive  sublimate, 
is  one  of  the  most  potent  germicides  known.  Exposure  for  half 
an  hour  to  a  solution  of  1  part  to  2000  parts  of  water  in  the  absence 
of  organic  material,  or  1  part  to  1000  if  it  be  present,  is  ample  to 
destroy  all  vegetative  forms.  Spores  are  killed  in  1  to  500  solu- 
tion in  one  hour.  The  value  of  bichloride  of  mercury  is  somewhat 
limited  by  the  fact  that  it  is  irritating  to  the  skin,  that  in  the  pres- 
ence of  alkaline  albuminous  substances  it  forms  inert  compounds, 


20  BACTERIOLOGY   FOR   NURSES 

and  that  it  has  a  corrosive  effect  upon  metals.  Its  use  then  as  a 
disinfectant  for  sputa,  feces,  etc.,  is  not  advisable  and  care  should 
be  exercised  in  using  it  about  household  plumbing. 

Potassium  Permanganate  ranks  high  as  a  germicide  for  certain 
purposes ;  its  greatest  usefulness  is  probably  in  surgical  practice. 
A  1  to  800  solution  will  kill  vegetative  forms.  Koch  found  that  a 
1  to  20  solution  killed  spores  in  one  day.  Its  application  is  some- 
what limited  on  account  of  the  readiness  with  which  it  combines 
with  organic  material  and  thus  becomes  inert. 

Sodium  Hydroxide  (Caustic  Soda) .  —  Vegetative  forms  of  bac- 
teria are  killed  in  a  few  minutes  by  a  1  per  cent  solution.  Spores 
are  killed  in  about  three  quarters  of  an  hour  in  a  4  per  cent  solution. 

Sodium  Carbonate  (Washing  Soda).  A  5  per  cent  solution  will 
destroy  vegetative  forms  in  a  few  hours.  It  has  no  effect  upon 
spores  at  ordinary  temperatures. 

Sodium  Bicarbonate  has  practically  no  germicidal  properties. 

CALCIUM   COMPOUNDS 

Slaked  Lime.  —  Lime  is  one  of  the  best  and  cheapest  disin- 
fectants known.  Slaked  lime  or  calcium  hydroxide  is  prepared 
by  adding  one  pint  of  water  to  two  pounds  of  lime.  A  1  per  cent 
solution  of  this  freshly  slaked  lime  will  kill  all  vegetative  bacteria 
within  a  few  hours.  A  3  per  cent  solution  kills  typhoid  bacilli 
in  one  hour.  Feces  or  other  infected  material  may  be  completely 
disinfected  if  mixed  with  equal  parts  of  a  20  per  cent  solution  and 
allowed  to  remain  in  contact  for  two  hours.  Freshly  slaked  lime 
should  always  be  used ;  if  left  exposed  to  the  atmosphere  it  absorbs 
more  water  and  carbon  dioxide  and  is  converted  into  calcium 
carbonate  (marble),  which  is  quite  inert. 

Milk  of  Lime  is  slaked  lime  diluted  with  four  to  eight  times 
its  volume  of  water.  It  should  not  be  used  if  more  than  a  few 
days  old  unless  well  protected  from  the  air. 

Chlorinated  Lime,  popularly  known  as  Chloride  of  Lime  or 
Bleaching  Powder,  may  be  used  either  as  a  dry  powder  or  in 
solution.  In  the  dry  state  it  is  frequently  used  in  privy  vaults 


DISINFECTANTS  21 

where  it  also  acts  as  a  deodorant  and  desiccant.  Under  certain 
circumstances  it  is  one  of  the  most  powerful  germicides  known. 
One  per  cent  solution  will  kill  most  bacteria  in  one  to  five  min- 
utes. A  5  per  cent  solution  usually  kills  all  spores  within  an 
hour.  Unfortunately  it  bleaches  and  destroys  fabrics. 

Recently,  chlorinated  lime  or  chlorinated  soda  has  come  into 
prominence  as  a  disinfectant  of  drinking  water.  Very  minute 
quantities  will  render  a  comparatively  clean  water  sterile. 

Labarraque's  Solution  contains  several  chlorine  compounds, 
chiefly  sodium  hypochlorite  and  sodium  chloride.  It  is  a  little 
more  expensive  and  not  so  efficient  as  chlorinated  lime. 

Antiformin  consists  of  a  solution  of  caustic  soda  and  chlorinated 
lime.  It  is  a  strong  germicide ;  a  2  to  5  per  cent  solution  will  kill 
most  vegetative  forms  in  five  minutes.  The  tubercle  bacillus  and 
other  members  of  the  same  group,  however,  are  slightly  affected 
by  it. 

Chlorine  Gas  is  strongly  germicidal.  Its  activity  is  due  to  the 
fact  that  in  the  presence  of  moisture  it  combines  with  the  hydro- 
gen, thus  liberating  oxygen  which  in  its  nascent  state  combines 
with  the  albuminous  substance  of  the  bacterial  cell.  A  0.2  per 
cent  watery  solution  kills  spores  in  5  minutes  and  the  vegetative 
forms  almost  immediately. 

Iodine  has  much  the  same  value  as  chlorine  both  in  its  gaseous 
state  and  in  solution. 

Acids  are  effective  germicides.  A  1  to  500  solution  of  sulphuric 
acid  kills  most  vegetative  forms  in  an  hour.  Hydrochloric  acid 
is  somewhat  weaker;  acetic,  citric,  and  salicylic  acids  are  much 
weaker  still.  A  2  per  cent  boric  acid  solution  will  destroy  the 
less  resistant  forms  of  bacteria. 

Sulphur  Dioxide  is  a  strong  insecticide  but  is  of  little  value 
as  a  germicide.  The  slight  action  it  possesses  on  bacteria  depends 
on  the  presence  of  moisture.  It  has  no  effect  upon  spores. 

Application  of  Disinfectants.  —  The  most  effective  place  to 
apply  disinfectants  is  as  near  the  seat  of  the  origin  of  infection  as 
possible.  As  the  excretions  from  the  mouth,  nose,  and  bowels, 
as  well  as  discharges  from  eruptions  or  wounds,  are  mainly  respon- 


22  BACTERIOLOGY   FOR   NURSES 

sible  for  the    spread  of  disease    their  immediate  disinfection  is 
imperative. 

RESULTS  OF  BACTERIAL  GROWTH 

Not  only  are  bacteria  influenced  by  their  surroundings,  but  they 
in  turn  exert  a  tremendous  influence  on  these  surroundings.  The 
effects  of  bacterial  growth  depend  largely  on  the  species  and  on  the 
composition  of  the  substance  on  which  they  happen  to  be  growing. 

Light.  —  Twenty  different  species  of  bacteria  have  been  de- 
scribed which  have  the  power  of  emitting  light.  The  phosphor- 
escence sometimes  seen  on  decaying  meat  and  fish  is  due  to  the 
growth  of  these  organisms.  They  are  widely  distributed  in  nature 
and  grow  most  luxuriantly  in  media  rich  in  salt,  as  in  sea-water. 
It  is  supposed  that  the  phenomenon  is  caused  by  a  substance, 
photogen,  which  is  closely  combined  with  the  cell  substance. 

Heat.  —  The  elevation  of  temperature  in  substances  undergoing 
bacterial  decomposition,  such  as  tobacco,  manure,  etc.,  is  often 
attributed  to  bacteria.  It  is  more  generally  supposed  that  the 
heat  is  due  to  chemical  reactions. 

Pigment.  —  When  bacteria  growing  on  culture  media  have 
become  sufficiently  numerous  to  be  seen  with  the  naked  eye,  they 
appear  as  a  more  or  less  moist  grayish  mass.  Certain  species, 
however,  have  the  ability  to  produce  a  pigment  which  may  give 
to  this  mass  a  brilliant  hue.  According  to  the  species  the  color 
may  be  violet,  blue,  red,  green,  orange,  or  yellow.  The  nature  of 
the  pigments  or  the  purpose  they  serve  are  not  fully  understood ; 
they  are  generally  thought  to  be  related  to  the  lipochromes,  the 
coloring  matter  met  with  in  the  yolk  of  eggs,  carrots,  etc. 

Chemical  Effect.  —  As  bacteria  consume  the  material  which 
serves  them  as  food  they  break  up  the  complex  organic  molecules 
into  simpler  ones  and  thus  entirely  change  the  chemical  nature  of 
their  surroundings.  Three  different  forms  of  bacterial  activity 
may  occur : 

1.  The  nourishment  of  the  cell  protoplasm  itself. 

2.  Excretion  from  the  bacterial  cell  of  waste  products. 

3.  Production  of  secretions  of  the  nature  of  ferments  or  enzymes,  which 

disintegrate  the  food  substance  on  which  they  are  growing. 


RESULTS   OF   BACTERIAL   GROWTH  23 

The  enzymes  produced  by  bacteria  are  probably  responsible 
for  all  the  processes  of  disintegration  to  which  they  give  rise. 
Two  general  forms  are  recognized :  fermentation  and  putrefaction. 
The  term  fermentation  is  usually  applied  to  the  breaking  down 
of  carbohydrates  into  alcohol,  acids,  carbon  dioxide,  etc.  Putre- 
factive decomposition  is  the  term  applied  to  the  breaking  down 
of  nitrogenous  substances.  It  is  generally  thought  that  bacterial 
action  on  proteins  closely  resembles  that  of  the  digestive  enzymes 
of  the  animal  alimentary  tract.  The  proteins  are  decomposed 
into :  proteoses,  peptones,  amino-acids,  etc.,  often  with  end- 
products  such  as  skatol,  mercaptan,  and  sulphureted  hydrogen. 
Ordinarily  these  processes  are  carried  on  by  aerobic  and  anaerobic 
bacteria  living  in  symbiosis. 


CHAPTER  III 

STERILIZATION  OF    GLASSWARE.     PREPARATION  OF 
CULTURE  MEDIA 

IN  order  to  become  familiar  with  the  characteristics  of  any  given 
species  of  bacteria,  it  is  necessary,  first  of  all,  to  isolate  it  from  every 
other  form.  In  nature  it  is  seldom  that  one  species  is  found  grow- 
ing alone.  This  does  occur  in  certain  diseases,  but  generally 
speaking  bacteria  have  to  be  taken  from  their  natural  surroundings 
and  grown  on  artificial  food  medium  in  order  to  be  studied. 

The  first  essential  then  for  bacterial  study  is  to  provide  condi- 
tions whereby  one  species  may  grow,  without  an  admixture  of 
other  forms.  Since  bacteria  are  practically  everywhere  this  can 
only  be  accomplished  by  first  destroying  all  germs  in  the  food 
medium  on  which  the  organism  is  to  be  cultivated  and  on  all  appa- 
ratus likely  to  come  in  contact  with  it.  In  order  to  do  this  one  form 
or  another  of  sterilization  is  employed,  the  method  used  depending 
largely  upon  the  object  to  be  sterilized ;  the  underlying  principle 
of  each,  however,  is  the  destruction  of  bacteria  by  heat.  Hot  air 
or  "  dry  heat  "  is  generally  used  for  glassware  and  hot  water  or 
steam,  "  moist  heat,"  for  culture  media. 

Cleaning  of  Glassware.  —  Before  sterilization  each  article  of 
glassware  should  be  perfectly  clean.  New  glass  as  a  rule  only 
requires  washing  with  soap  and  water  and  the  adherent  dirt  re- 
moved with  a  test-tube  brush.  Old  glassware  containing  cul- 
tures should  be  sterilized  either  in  the  autoclave  or  boiled  in  5 
per  cent  solution  of  washing  soda  or  soapsuds  for  one  hour  in  a 
covered  boiler.  Glassware  may  be  further  cleansed  if  necessary 
by  placing  for  one  hour  or  more  in  the  following  chromic  acid 
mixture : 

24 


STERILIZATION   OF   GLASSWARE  25 

Bichromate  of  potassium 6  parts 

Water 100  parts 

Sulphuric  acid 6  parts 

Dissolve  the  bichromate  by  heating  in  an  agate  kettle.  Add 
the  sulphuric  acid  slowly  on  account  of  the  heat  generated ;  after 
cooling  keep  in  a  glass  jar.  The  mixture  may  be  used  more  than 
once. 

After  thoroughly  rinsing  and  drying,  test  tubes  and  flasks  are 
plugged  with  ordinary  non-absorbent  cotton.  The  plugs  should 
not  be  twisted  or  creases  will  form,  making  possible  the  entrance  of 
bacteria  from  the  surrounding  air.  The  most  satisfactory  method 
is  to  roll  the  cotton,  which  should  fit  just  tight  enough  to  allow 
one  to  lift  the  tube  by  means  of  the  plug. 


FIG.  5.  —  Hot  Air  Sterilizer. 

STERILIZATION   BY  DRY  HEAT 

1.  Hot  Air  Chamber.  —  This  method  is  used  for  all  forms  of 
glassware.  The  hot  air  chamber  (Fig.  5)  consists  of  an  outer 
and  an  inner  covering  of  sheet  iron.  At  the  bottom,  in  the  space 
between  the  two  jackets,  several  gas  jets  are  arranged.  The 


26 


BACTERIOLOGY    FOR   NURSES 


air  thus  heated  surrounds  the  inner  chamber  and  escapes  through 
holes  in  the  top  of  the  outer  case.  The  bulb  of  the  thermometer 
used  to  record  the  temperature  should  pass  about  to  the  center 
of  the  inner  chamber.  Heating  for  one  hour  at  150°  C.  is  sufficient 
to  insure  sterilization.  Articles  should  be  placed  in  the  hot  air 
chamber,  without  crowding,  before  heating  it  and  should  be  allowed 

to  remain  there  after  sterilization  is 
complete  until  the  temperature  falls. 
Sudden  heating  or  cooling  may  cause 
the  glass  to  crack. 

2.  Red  Heat.  —  Platinum  needles  are 
sterilized  by  flaming  in  a  Bunsen  burner 
until  they  are  red  hot.  The  points  of 
forceps,  coverlips,  and  glass  slides  may 
also  be  sterilized  by  passing  through  a 
Bunsen  flame. 

STERILIZATION  BY  MOIST  HEAT 

1.  Boiling. — The  boiling  of  water  for 
five  minutes  is  sufficient  to  insure  steri- 
lization if  no  spores  are  present.  Steel 
instruments  should  be  placed  in  a  boiling 
solution  of  1  per  cent  sodium  carbonate 
for  at  least  five  minutes. 

2.  Steam  at  High  Pres- 
sure. —  The  most  rapid  and 
effective  means  of  steriliza- 
tion is  accomplished  by  steam 
under  pressure  in  an  autoclave  (Fig.  6).  The  apparatus  consists 
of  a  metal  cylinder  supported  in  an  iron  case ;  it  is  fitted  with  a 
pressure  gauge,  thermometer,  and  safety  valve ;  its  cover  can  be 
securely  fastened  down  with  nuts  and  screws.  The  articles  to 
be  sterilized  are  supported  on  a  perforated  diaphragm,  and  heat 
to  generate  the  steam  is  supplied  by  a  large  Bunsen  burner 
beneath.  The  temperature  usually  employed  is  115°  C.  to  120° 
C.  In  order  to  obtain  a  temperature  of  115°  C.  a  pressure  of 


FIG.  6.  —  Autoclave. 


STERILIZATION  OF  GLASSWARE  27 

about  23  pounds  to  the  square  inch  is  necessary,  that  is,  8  pounds 
more  than  the  ordinary  atmospheric  pressure  of  15  pounds.  To 
reach  120°  C.  the  pressure  must  mount  to  30  pounds  or  15  pounds 
plus  the  atmospheric  pressure.  Temperature  and  pressure  corre- 
spond thus: 

Temperature  about  115°  C.  —  Increased  pressure  necessary,  8  Ib. 
Temperature  about  120°  C.  —  Increased  pressure  necessary,  15  Ib. 

A  temperature  of  120°  C.  maintained  for  15  minutes  is  sufficient 
to  sterilize  media  in  test  tubes.  Media  in  bulk  is  generally  allowed 
30  minutes. 

In  using  the  autoclave  care  should  be  taken  (1)  that  baskets  of 
tubes  are  not  placed  one  on  the  top  of  the  other  or  the  plugs  will 
become  wet  from  the  dripping.  (2)  All  air  should  be  displaced  by 
steam  before  closing  the  stopcock.  If  a  mixture  of  air  and  steam 
is  present  the  exact  temperature  is  not  recorded.  (3)  The  pressure 
should  be  allowed  to  drop  to  zero  before  opening  the  stopcock; 
a  too  rapid  removal  of  the  pressure  may  cause  the  fluid  media  to 
be  blown  out  of  the  tubes. 

The  autoclave  is  used  mainly  for  the  sterilization  of  sugar- 
free  media  and  discarded  cultures.  As  gelatin,  blood  serum, 
and  all  media  containing  sugars  are  changed  chemically  when 
heated  for  a  long  time  at  a  high  temperature,  they  are  sterilized 
by  another  method. 

Discontinuous  Sterilization,  sometimes  spoken  of  as  intermittent 
or  fractional  sterilization.  There  are  two  forms  of  applying  this 
method : 

1.  Heating  in  steam  at  100°  C.  for  20  minutes  on  each  of  six  successive 

days. 

2.  Heating  in  steam  from  56°  C.  to  70°  C.,  1  hour  on  each  of  three  suc- 

cessive days. 

The  former  method  is  used  for  gelatin  and  all  sugar  media  that 
would  be  injured  by  autoclave  sterilization;  the  latter  method 
is  employed  for  media  containing  blood  serum  or  transudates 
from  body  cavities,  such  as  ascitic  fluid. 

The  principle  underlying  both  methods  is :  all  bacteria  when 
free  from  spores  are  killed  by  the  temperature  of  boiling  water, 


28 


many  even  at  a  much  lower  temperature.  Thus  heating  on  the 
first  day  will  kill  all  non-spored  forms.  During  the  twenty-four 
hours'  interval  the  media  is  kept  at  22°  C.  in  order  that  spores  if 
present  may  develop  into  vegetative  forms  and  be  killed  during 
the  second  heating.  In  case  any  of  the  spores  have  been  slow  in 
developing  and  thus  escaped  the  second  heating  the  process  is 

repeated  a  third  time.  Generally 
speaking  this  method  gives  good 
results ;  it  is  advisable,  however, 
to  prolong  the  heating  of  media 
in  flasks  to  half  an  hour. 

The  "Arnold  "  steam  sterilizer 
(Fig.  7)  has  almost  entirely  dis- 
placed that  introduced  by  Koch 
for  this  form  of  sterilization.  It 
is  constructed  with  a  false  bottom 
so  that  a  small  quantity  of  water 
may  be  heated  to  produce  steam 
quickly.  A  perforated  tin  dia- 
phragm permits  the  steam  to 
stream  up  and  surround  the  ob- 
jects placed  on  it.  As  the  steam 
rises  it  passes  through  an  opening 
in  the  top  of  the  inner  jacket 
and  descends  as  water  of  con- 
densation to  again  feed  the  water 
below.  By  this  method  as  by 
that  of  autoclave  sterilization 
no  evaporation  takes  place  from 

the  media  because  it  is  surrounded  with  an  atmosphere  already 
saturated  with  moisture. 

An  ordinary  kitchen  steamer  over  a  pot  of  boiling  water  may  be 
used  if  an  Arnold  sterilizer  is  not  available. 

For  the  second  method,  i.e.  heating  at  a  lower  temperature 
on  six  consecutive  days,  an  inspissator  (Fig.  8)  is  generally 
used. 


FIG.  7.  —  Arnold  Sterilizer. 


PREPARATION  OF  CULTURE  MEDIA 


29 


Rubber  Stoppers  and  Tubing  should  never  be  heated,  they 
should  be  cleansed  with  soap  and  water  and  allowed  to  stand  for 
one  hour  in  a  1  to  1000  solution  of  bichlorid  of  mercury,  then 
washed  with  sterile  water  before  using. 


CULTURE  MEDIA 

One  of  the  points  to  be  observed  in  the  artificial  culture  of  bac- 
teria is  that  the  food  medium  supplied  should  resemble  as  closely  as 
possible  that  to  which  the 
organism  is  accustomed. 
Many  species  grow  luxu- 
riantly if  only  a  simple 
nitrogen  and  carbon  com- 
pound and  some  salt  and 
water  are  present;  others 
require  more  complex 
substances.  For  certain 
pathogenic  bacteria  body 
fluids  such  as  blood  serum 
and  ascitic  fluid  are  used. 
The  fact  that  certain  spe- 
cies will  grow  abundantly 
on  one  kind  of  medium  and  not  at  all  on  another,  or  that  char- 
acteristic growth  will  occur  on  certain  media,  is  often  an  aid  in 
the  identification  of  species. 

The  most  commonly  used  media  have  as  their  basis  a  watery 
extract  of  meat  to  which  a  small  amount  of  peptone  is  added. 
Koch  found  that  by  the  addition  of  gelatin  to  this  broth  a  solid 
transparent  medium  would  result  on  which  growth  from  a  single 
organism  could  be  obtained  and  on  which  also  certain  bacterial 
characteristics  became  evident.  Gelatin,  however,  has  only  a 
limited  use;  it  does  not  remain  solid  at  the  temperature  best 
suited  for  pathogenic  microorganisms ;  moreover,  there  are  certain 
species  of  bacteria  which  during  their  growth  are  able  to  liquefy 
it  at  lower  temperatures.  To  obviate  these  conditions  agar, 


FIG.  8. —  Inspissator. 


30  BACTERIOLOGY  FOR  NURSES 

a  product  derived  from  the  stems  of  seaweed  growing  in  the 
Chinese  seas,  has  been  substituted.  Agar  is  not  liquefied  by  the 
action  of  bacteria,  does  not  melt  below  98°  C.,  and  on  cooling 
solidifies  at  about  39°  C. 

Certain  technical  procedures  such  as  adjusting  the  reaction, 
clearing  and  filtering  of  media,  should  be  Understood  before  the 
preparation  is  attempted. 

Titration  and  Adjustment.  —  A  moderately  alkaline  reaction 
to  litmus  is  suitable  for  the  growth  of  most  pathogenic  bacteria. 
Generally  the  medium  mixture  is  at  first  too  acid;  it  may  be 
reduced  by  the  addition  of  4  per  cent  sodium  hydrate  solution  until 
red  litmus  paper  is  turned  slightly  blue  and  blue  litmus  retains 
its  color.  The  solution  must  be  well  stirred  into  the  media  and  the 
test  paper  immersed  in  the  liquid  ;  on  no  account  should  the  test- 
ing be  done  by  dropping  media  on  to  the  paper  by  means  of  a  glass 
rod. 

Litmus  is  not  a  delicate  indicator  and  if  a  more  accurate  adjust- 
ment is  required  a  more  complicated  method  is  adopted,  with 
phenolphthalein  as  an  indicator.  The  neutral  points  are  different  ; 
media  alkaline  to  litmus  may  be  still  acid  to  phenolphthalein. 

The  standard  method  for  titration  is  as  follows:  5  c.c.  of 
medium  and  45  c.c.  of  distilled  water  are  mixed  in  a  casserole 
and  boiled  for  one  minute,  then  1  c.c.  of  the  phenolpthalein  solution 
(0.5  per  cent  in  50  per  cent  alcohol)  is  added.  If  no  color  appears 
the  medium  is  acid,  and  while  hot  twentieth  normal  sodium  hydrox- 


ide  solution  f  NaOH  —  J  is  run  into  the  casserole  from  a  burette 
\  ZO/^ 

until  a  faint  but  distinct  color  is  seen.  This  color  must  remain 
on  stirring,  otherwise  more  alkali  is  needed.  From  the  amount 
added  the  acidity  of  the  medium  is  determined  and  an  estimate 

(  N\ 

made  of  the  amount  of  normal  solution  f  NaOH  —  1  to  give  the 

required  reaction  to  the  bulk  of  the  medium  necessary. 

N 
For  example,  if  5  c.c.  of  medium  required  2.4  c.c.  of  ^  NaOH 

Z\J 

to  neutralize  it,  100  c.c.  (twenty  times  as  much)  would  require 


PREPARATION  OF  CULTURE  MEDIA     31 

N 
2.4  c.c.  of  —  NaOH  (twenty  times  as  strong) ;  in  other  words  the 

medium  is  2.4  per  cent  acid  to  phenolphthalein  (+2.4).  Assum- 
ing the  required  reaction  is  +1  per  cent  we  must  add  2.4  c.c. 

N 
minus  1  c.c.  or  1.4  c.c.  of  •—  NaOH  to  every  100  c.c.  of  medium,  or 

14  c.c.  to  a  liter.  The  plus  sign  is  used  to  indicate  an  acid  and 
the  minus  sign  an  alkaline  reaction  to  phenolphthalein. 

Should,  on  the  other  hand,  the  mixture  show  a  pink  color  when 

N 
the  indicator  is  added,  the  medium  is  alkaline  and  —  HC1  is  used 

£\J 

instead  of  the  sodium  hydroxide  solution  as  above,  until  only  a 
very  faint  color  persists.  If,  for  example,  0.5  c.c.  of  the  acid  has 
been  used  then  the  medium  is  0.5  per  cent  alkaline  (  —  0.5%)  and 

N 
it  will  require  0.5  c.c.  of  —  HC1  for  every  100  c.c.,  or  5  c.c.  for 

every  liter,  to  bring  it  to  the  neutral  point.  If  the  required  reac- 
tion is  +1  or  1  per  cent  acid,  then  5  c.c.  plus  10  c.c.,  or  15  c.c.,  of 

N 

—  HC1  must  be  added  to  each  liter  of  medium. 

Clearing  Media.  —  This  is  accomplished  as  follows :  One  or 
two  eggs  for  each  liter  of  medium  are  lightly  beaten  in  a 
pan  and  mixed  with  a  little  water.  The  medium  is  cooled  to 
below  60°  C.  and  the  eggs  are  thoroughly  stirred  into  it.  The 
mixture  is  then  heated  in  the  Arnold  Sterilizer  or  autoclave 
and  as  the  egg  albumin  coagulates,  it  enmeshes  and  carries 
down  with  it  as  a  sediment  all  the  fine  particles,  thus  leaving 
the  medium  clear. 

Filtering  Media.  —  Fluid  media  may  be  filtered  through  paper ; 
for  media  which  will  solidify  on  cooling  absorbent  cotton  is  better. 
In  the  latter  case  a  spiral  of  copper  wire  is  placed  in  the  bottom 
of  the  funnel  and  two  moderately  thick  squares  of  absorbent 
cotton  are  so  arranged  over  it  that  the  fibers  of  one  are  at  right 
angles  to  those  of  the  other.  Paper  or  cotton  should  first  be  mois- 
tened with  water  so  that  any  fat  in  the  media  will  not  pass  through. 
The  first  filtrate  may  not  be  clear,  in  which  case  it  should  be  passed 


32 


BACTERIOLOGY  FOR  NURSES 


through  the  filter  a  second  time.     Media  which  solidify  when 
cool  should  be  filtered  in  a  warm  place. 

Tubing  of  Media.  —  For  the  most  part  media  are  used  in  test 
tubes.  An  improvised  apparatus  (Fig.  9)  may  be  arranged  for 
filling  them  as  follows :  A  piece  of  rubber  tubing  is  attached  at 
(one  end  to  a  glass  funnel  and  at  the  other  to  a  glass  point;  a 
pinchcock  is  fixed  at  the  center  of  the  tube.  The  plug  is  removed 
from  the  tube  by  taking  it  between  the  third  and  fourth  fingers 

of  the  right  hand  and  the  glass 
point  is  placed  almost  to  the  bot- 
tom of  the  test  tube  in  order  to 
prevent  the  medium  touching  the 
neck  of  the  tube  where  the  cotton 
plug  might  stick.  About  7  c.c. 
of  medium  is  sufficient  for  each 
tube.  The  next  step  is  steriliza- 
tion by  the  method  most  suitable, 
after  which  the  medium  is  ready 
for  use. 

Preparation  of  Culture  Media 
in  Common  Use.  —  The  basis  of 
the  most  commonly  used  media 
is  an  infusion  of  meat,  or  meat 

FIG.  9.  —  Apparatus  for  tubing  Media.  t_«  r  11 

extract,  to  which  a  small  amount 
of  peptone  and  sodium  chloride  is  added. 

Meat  infusion  is  prepared  as  follows:  One  liter  of  water  is 
poured  over  one  pound  of  finely  chopped  beef  or  veal.  It  is  heated 
at  a  temperature  of  50°  C.  for  one  hour  or  it  may  be  allowed  to 
remain  twenty-four  hours  in  the  refrigerator  and  not  heated. 
The  infusion  is  then  strained  through  cheesecloth  and  all  the  juice 
thoroughly  pressed  out  of  the  meat.  The  fluid  contains  soluble 
albumins,  extractives,  muscle  sugar,  and  salts. 

As  a  substitute  for  meat  infusion  Liebig's  extract  2  to  3  grams 
per  liter  of  water  may  be  used. 


PREPARATION  OF  CULTURE  MEDIA  33 

1.  Nutrient  Broth. 

Meat  infusion  or  meat  extract 1000  c.c. 

Peptone 10  gm. 

Sodium  chloride 5  gm. 

Warm  the  meat  infusion  to  50°  C.,  add  the  peptone  and  salt  and 
stir  until  the  peptone  is  dissolved.  Add  a  little  sodium  hydrox- 
ide to  reduce  the  acidity,  boil  to  coagulate  the  albumin  present ; 
ordinarily  it  is  not  necessary  to  clear  with  eggs.  Add  water  to 
make  up  for  that  lost  by  evaporation,  adjust  reaction,  filter,  and 
tube  or  place  in  flasks  for  sterilization.  Nutrient  broth  is  of 
service  in  obtaining  the  soluble  toxins  formed  by  bacteria  and  in 
determining  motility. 

2.  Glucose  Broth.  —  1  or  2  per  cent  glucose  is  added  to  nutrient 
broth.     The  procedure  is  the  same  as  in  (1)  except  sterilization 
should  be  by  the  fractional  method.     Glucose  is  a  reducing  agent, 
consequently  no  free. oxygen  can  remain  in  a  medium  containing 
it.     Glucose  broth  on  this  account  is  used  for  the  cultivation  of 
anaerobic  organisms. 

3.  Glycerin  Broth.  —  To  broth  (1)  after  filtration,  5  to  8  per 
cent  of  glycerin  is  added.     This  medium  is  used  especially  for 
growing  the  tubercle  bacillus  when  tuberculin  is  to  be  prepared. 

4.  Gelatin  Medium. 

Meat  infusion  or  extract         1000  c.c. 

Peptone 10  gm. 

Sodium  chloride 5  gm. 

Gelatin  (gold  label) 100  gm. 

This  is  simply  the  above  broth  with  the  addition  of  gelatin  as  a 
solidifying  agent.  The  ingredients  are  dissolved  by  warming, 
the  reaction  is  adjusted,  eggs  are  added  to  clear  the  medium 
(page  19),  and  it  is  then  heated  for  15  minutes.  Water  is  added 
to  make  up  the  original  volume;  after  being  thoroughly  stirred 
the  medium  is  filtered  through  cotton,  tubed,  and  sterilized  by 
fractional  sterilization.  Characteristic  growth  takes  place  on 
gelatin  media  which  often  facilitates  identification. 

5.  Agar  Medium. 

Nutrient  broth  (1) 1000  c.c. 

Shredded  agar 15  c.c. 

D 


34 


BACTERIOLOGY  FOR  NURSES 


The  agar  is  added  to  the  broth  and  dissolved  by  boiling  the  mix- 
ture for  thirty  to  forty-five  minutes.  Loss  by  evaporation  is  made 
up  by  the  addition  of  water,  the  reaction  is  adjusted,  and  the  media 
cleared,  heated,  filtered,  tubed,  and  sterilized  in  the  autoclave. 
Agar  medium  serves  a  great  variety  of  purposes ;  it  is  perhaps  the 
most  frequently  used  of  all  media. 

6.  Potato  Medium.  —  Large  potatoes  are  chosen,  thoroughly 
scrubbed  with  a  brush  and  then  peeled,  after  which  they  are  kept 

under  running  water  to  prevent  discoloration. 
Cylindrical  pieces  are  removed  by  means  of  a 
large  apple  corer,  and  the  cylinders  in  turn  are 
cut  in  half  diagonally. 

The  reaction  of  the  potato  is  normally  acid. 
This  is  corrected  by  leaving  the  pieces  over- 
night in  running  water  or  by  placing  them  in  a 
•k  I'  1  per  cent  solution  of  sodium  carbonate  for  half 
ML  an  hour.  Each  cylinder  is  placed  in  a  large  test 
155^  tube  with  the  slant  surface  uppermost  (Fig.  10). 
About  one  c.c.  of  water  may  be  added  to  retard 
drying  and  the  tubes  sterilized  by  the  fractional 
method.  Potato  is  generally  chosen  as  a  medium  when  pigment 
production  is  to  be  studied. 

7.  Glycerin  Potato  is  prepared  by  covering  the  potato  slices 
in  the  tube  with  a  6  per  cent  solution  of  glycerin  in  water  and 
steaming  in  the  Arnold  Sterilizer  for  half  an  hour.     The  glycerin 
is  then  poured  off  and  the  tubes  are  sterilized  for  another  half 
hour.     Glycerin  potato  is  sometimes  used  for  the  cultivation  of 
the  tubercle  bacillus. 

8.  Peptone  Water. 


FIG.  10.— Potato 
Tube. 


Water        .     .     . 
Peptone     .     . 
Sodium  chloride 


100  c.c. 
2  c.c. 
0.5  c.c. 


The  peptone  and  salt  are  dissolved  in  the  water  by  heating. 
As  the  fluid  is  generally  alkaline  it  does  not  need  adjusting 
unless  required  for  special  purposes;  it  may  be  filtered,  tubed, 
and  sterilized  at  once.  Peptone  water  is  used  to  test  for  the 


PREPARATION  OF  CULTURE  MEDIA 


35 


formation  of  indol ;  it  is  also  used  as  a  basis  for  other  special 
media. 

Indicators.  —  To  any  of  the  ordinary  media,  substances  may  be 
added  which  serve  to  show  any  difference  in  reaction  taking  place 
during  bacterial  growth  ;  in  other  words,  they  indicate  the  ability 
of  the  microorganism  present  to  produce  fermentative  or  putre- 
factive changes.  Litmus  is  perhaps  the  most  generally  used. 
After  filtration  of  the  medium,  which  should  be  slightly  alkaline, 
sufficient  of  a  reliable  solution  such  as  the  "  Kubel-Tiemann  Solu- 
tion "  is  added  to  give  a  distinctly  bluish  tint.  A  deepening  of 
the  blue  color  will  indicate  increased  alkalinity;  a  change  from 
blue  to  a  pink  color  will  reveal  the  presence  of  an  acid.  Neutral 
red  is  an  indicator  frequently  used;  in  the  presence  of  acid  it 
becomes  a  deep  rose  color, 
in  an  alkaline  medium  it  is 
yellow  with  sometimes  a 
green  fluorescence. 

9.  Milk.  —  Fresh  milk  is 
placed  overnight  in  the  ice 
box  so  that  the  cream  may 
rise ;     in  the    morning   the 
milk  is  siphoned  off  from  be- 
neath the  cream  and  sufficient  litmus  added  to  give  it  a  purplish 
blue  color.     It  will  generally  be  found  to  be  alkaline ;  if  not,  a  little 
sodium  hydroxide  solution  should  be  added  to  give  it  the  required 
color.  It  is  then  ready  for  tubing  and  fractional  sterilization.  Litmus 
milk  is  a  convenient  medium  for  observing  the  ability  of  certain  bac- 
teria to  ferment  lactose,  or  to  coagulate  the  soluble  albumin  present. 

10.  Neutral-Red  Lactose  Peptone  Medium. 

Peptone  solution    (8) 100  c.c. 

Lactose 1  gm. 

Saturated  aqueous  solution  of  neutral  red      ...         1  c.c. 

The  medium  is  filtered  into  fermentation  tubes  (Fig.  11)  and  steri- 
lized by  the  fractional  method.  This  medium  is  used  largely  for 
the  examination  of  water  and  shellfish  to  determine  by  the  presence 
of  the  colon  bacillus  whether  sewage  pollution  has  occurred. 


FIG.  11. —  Fermentation  Tubes. 


36  BACTERIOLOGY  FOR  NURSES 

11.  Conradi  Drigalsky  Medium. 

(a)  Agar 20  gm..  (6)  130  gm.  of  Kubel  and  Tiemann's 

Sodium  chloride     .     .  5  gm.  litmus  solution 

Peptone 20  gm.  10  c.c.  of  1  to  1,000  solution 

Nutrose 10  gm.  of  crystal  violet 

Liebig's  Extract     .     .  4  gm.  15  gm.  lactose 

Water 1000  c.c. 

The  ingredients  (a)  should  be  thoroughly  mixed  and  heated  in 
the  autoclave  to  dissolve.  The  mixture  should  then  be  cooled  to 
below  60°  C.,  cleared,  sufficient  sodium  hydroxide  solution  added 
to  give  a  decided  alkaline  reaction  to  litmus,  and  then  filtered. 
Ingredients  (6)  are  next  added  and  the  medium  heated  for  10 
minutes  in  the  Arnold  in  order  to  obtain  a  thorough  mixing.  It 
should  then  be  tubed  and  sterilized  by  the  fractional  method. 

This  is  one  of  the  media  used  specially  for  the  isolation  of  the 
typhoid  group  of  organisms;  the  principle  being  that  while  the 
food  supply  is  favorable  to  the  typhoid  and  colon  bacilli  the  growth 
of  other  intestinal  bacteria  is  inhibited  by  the  antiseptic  action 
of  the  crystal  violet.  The  difference  between  the  colonies  formed 
by  the  colon  bacilli  and  those  formed  by  the  typhoid  bacilli  is 
readily  seen  in  the  plated  medium.  The  B.  coli  colonies  are  dis- 
tinctly red  and  non-transparent,  while  those  of  B.  typhosus  are 
smaller,  bluish  violet  in  color  and  of  a  somewhat  glassy  appearance. 

12.  Loeffler's  Serum.  —  Three  parts  of  calf  or  sheep  serum  is 
mixed  with  one  part  of  nutrient  broth  (1)  which  has  been  made 
neutral  to  litmus.     One  per  cent  dextrose  is  added  and  the  medium 
is  tubed.     In  order  to  coagulate  the  serum  the  tubes  are  placed 
in  an  inspissator  in  a  slanting  position,  or  an  Arnold  sterilizer  may 
be  used  if  the  outer  cover  is  replaced  by  a  cloth  and  the  temper- 
ature is  allowed  to  rise  very  gradually  to  80°  C.     After  coagulation 
the  medium  is  sterilized  by  the  fractional  method.     This  medium 
is  especially  suitable  for  the  growth  of  the  diphtheria  bacillus ;  it 
is  useful  also  for  other  organisms. 

13.  Blood  Agar.  —  One  c.c.  of  fresh  or  defibrinated  blood  is 
added  to  about  6  c.c.  of  melted  agar  cooled  to  42°  C.  to  45°  C., 
well  mixed  and  either  slanted  in  the  tube  or  poured  into  a  Petri 
dish.     The  simplest  method  of  preparation  is  to  thoroughly  cleanse 


PREPARATION  OF   CULTURE  MEDIA  37 

a  finger  and  then  wash  with  alcohol,  allow  the  alcohol  to  evaporate 
and  then  with  a  sterile  needle  prick  the  finger.  Take  up  a  drop 
of  the  blood  with  a  sterile  platinum  loop  and  smear  it  on  the  sur- 
face of  an  agar  slant.  Agar  poured  out  in  a  thin  layer  in  a  Petri 
dish  may  be  smeared  with  blood  in  the  same  way  and  used  for 
cultures.  It  is  advisable  to  incubate  the  blood-smeared  medium 
for  24  hours  before  inoculating  to  be  sure  that  it  is  sterile. 

Organisms  such  as  the  gonococcus,  pneumococcus,  and  influ- 
enza bacillus,  which  do  not  grow  readily  on  ordinary  agar,  are  culti- 
vated on  this  medium. 

14.  Hiss  Serum  Water.  —  Beef  serum  is  drawn  in  a  pipette 
from  clotted  blood  and  added  to  distilled  water  in  the  proportion 
of  one  part  of  serum  to  two  or  three  parts  of  wrater.  The  mixture 
is  heated  in  the  Arnold  for  15  minutes  at  100°  C.  to  destroy  any 
sugar  fermenting  enzyme  present  in  the  serum,  after  which  sufficient 
aqueous  solution  of  litmus  is  added  to  give  a  transparent  blue  color. 
One  per  cent  of  the  desired  sugar  (glucose,  saccharose,  lactose,  etc.) 
is  added  and  the  medium  is  tubed  and  sterilized  by  the  fractional 
method  in  the  Arnold. 

This  medium  is  of  use  in  determining  the  ability  of  a  species  to 
ferment  different  carbohydrates  and  also  its  power  to  coagulate 
serum  protein. 

Method  of  Obtaining  Serum.  —  Beef  or  sheep's  blood  is  collected 
in  a  sterile  jar  at  the  slaughter  house.  After  coagulation  the 
clot  should  be  carefully  separated  from  the  sides  of  the  container 
with  a  sterile  glass  rod  and  the  jar  placed  in  the  refrigerator  for 
24  hours.  At  the  end  of  this  time  the  clear  serum  can  be  pipetted 
or  siphoned  off  with  sterile  glass  tubing  and  placed  in  sterile  flasks. 
Serum  may  be  sterilized  in  its  fluid  state  by  exposure  to  a  temper- 
ature of  60°  C.  for  one  hour  upon  six  consecutive  days. 

Exudates  from  the  pleural  or  abdominal  cavity  may  be  used 
instead  of  beef  or  sheep  serum.  The  fluid  is  allowed  to  flow  directly 
out  of  the  canula  into  sterile  flasks.  The  instruments  should  be 
taken  into  the  ward  in  sterile  water  and  not  in  an  antiseptic  solu- 
tion. Before  using  the  fluid  should  be  incubated  and  any  flasks 
showing  contamination  should  be  discarded. 


CHAPTER  IV 

MICROSCOPIC    EXAMINATION    AND     STAINING    OF 

BACTERIA 

The  Microscope.  —  In  order  to  study  the  structure  and  move- 
ments of  individual  bacteria  a  good  microscope  is  essential  (Fig. 
12).  A  complete  instrument  generally  has  four  oculars  or  eye  pieces 
A  numbered  from  1  to  4.  Number  one  gives  the  lowest  magnifi- 
cation and  number  four  the  highest.  At  the  lower  end  of  the 
tube  B  there  are  usually  three  objectives  C  attached  to  a  revolving 
nosepiece;  the  objectives  give  the  main  magnification.  For 
the  examination  of  groups  of  bacteria  growing  together  in  solid 
media,  when  one  wishes  to  see  only  the  general  appearance  of  the 
assemblage,  the  lowest  magnification  is  used,  i.e.  ocular  1  or  2  and 
objective  4.  For  unstained  preparations,  when  motility  or  serum 
reaction  is  to  be  studied,  ocular  2  or  3  and  objective  7  is  employed. 
When  the  finer  structures  of  individual  organisms  are  to  be  noted 
in  stained  preparations,  ocular  4  and  the  oil  immersion  objective 
T2  should  be  used;  the  latter  combination  gives  a  magnification 
of  about  one  thousand  diameters.  When  the  oil  immersion  lens 
is  employed  a  small  drop  of  oil  of  the  same  index  of  refraction  as 
the  glass  lens  (cedar  oil  is  generally  used)  is  placed  on  the  prepara- 
tion to  be  examined.  The  tube  is  lowered  until  the  objective  is 
connected  with  the  slide  by  means  of  the  oil,  thus  all  the  rays  of 
light  are  held  together  and  pass  into  the  tube  without  the  loss 
through  deflection  which  occurs  when  air  fills  the  intervening  space 
between  the  slide  and  the  dry  objective.  After  using,  the  oil 
should  be  gently  wiped  from  the  lens  with  Japanese  lens  paper 
or  a  clean  soft  linen  handkerchief.  If  absolutely  necessary  a  little 
zylol  may  be  used  but  never  alcohol ;  the  latter  dissolves  the  ma- 
terial by  means  of  which  the  lens  is  fixed  in  its  metal  container. 

38 


THE  MICROSCOPE 


39 


The  stage  D  serves  as  a  table  on  which  to  place  the  object  to 
be  examined.  Immediately  beneath  the  opening  in  the  stage  is 
the  Abbe  condenser  E,  a  system  of  lenses  which  serves  to  condense 
the  rays  of  light  passing  from  the  mirror  F  to  the  object  in  such  a 
way  as  to  give  the  greatest  luminosity  possible. 


FIG.  12.  —  Microscope. 

The  iris  diaphragm  G  is  placed  between  the  condenser  and  the 
mirror  and  serves  the  same  purpose  as  the  iris  of  the  eye  in  regulat- 
ing the  amount  of  light  admitted.  For  unstained  preparations, 
when  it  is  desired  to  bring  out  in  relief  the  margins  of  the  organisms 
to  be  studied,  a  small  aperture  of  the  diaphragm  is  used  in  conjunc- 
tion with  the  concave  mirror.  For  stained  preparations  the 


40  BACTERIOLOGY  FOR  NURSES 

diaphragm  should  be  widely  opened  and  the  flat  side  of  the  mirror 
used. 

By  means  of  the  coarse  adjustment  H  the  tube  of  the  microscope 
can  be  raised  or  lowered  ;  it  is  used  to  bring  the  object  to  be  studied 
roughly  into  focus.  The  fine  adjustment  I  raises  or  lowers  the 
tube  much  more  slowly  and  evenly  and  is  used  with  high-power 
objectives  in  order  to  obtain  a  clear  sharp  definition  of  the  object 
after  it  has  been  brought  into  focus  by  the  coarse  adjustment. 
The  fine  adjustment  has  a  limited  range  and  should  never  be  forced ; 
when  it  ceases  to  operate  the  tube  should  be  raised  by  means  of 
the  coarse  adjustment  and  the  screw  should  be  turned  back  mid- 
way within  its  range. 

To  focus,  lower  the  tube  by  means  of  the  coarse  adjustment 
until  the  objective  nearly,  but  not  quite,  touches  the  slide  to  be 
examined.  Then  with  the  eye  looking  through  the  ocular  raise 
the  tube  until  the  object  can  be  plainly  seen ;  when  it  is  well  in 
focus  use  the  fine  adjustment  to  bring  out  more  clearly  the  part 
of  the  field  to  be  studied.  In  focusing  with  the  coarse  adjustment 
care  should  be  taken  never  to  lower  the  tube  while  the  eye  is  look- 
ing through  the  ocular ;  if  the  light  is  too  intense  or  the  preparation 
transparent  the  focal  point  may  be  passed  without  being  perceived 
and  the  result  may  be  a  broken  slide  and  a  damaged  lens. 

Light.  —  The  best  light  for  microscopic  work  is  that  obtained 
from  white  clouds  or  blue  sky  with  a  northern  exposure;  direct 
sunlight  should  be  avoided.  Satisfactory  artificial  light  may  be 
obtained  by  means  of  a  Welsbach  burner  and  a  whitened  incan- 
descent bulb. 

Dark  Ground  Illumination.  —  An  apparatus  to  be  used  in  con- 
junction with  the  microscope  has  been  devised  whereby  minute 
particles  are  made  visible,  particles  which  could  not  be  seen  other- 
wise even  with  the  highest  magnification  obtainable.  The  general 
principle  involved  is  to  arrange  for  light  to  be  thrown  obliquely 
on  the  object  to  be  examined  and  to  stop  the  rays  passing  directly 
towards  the  tube  of  the  microscope.  An  electric  arc  lamp  is  used 
as  a  source  of  light ;  the  organisms  appear  as  brightly  illumined 
objects  while  the  fluid  which  surrounds  them  forms  a  dark  back- 


EXAMINATION   AND  STAINING   OF  BACTERIA    41 

ground.  The  method  may  be  employed  for  the  examination  of 
bacteria  in  general ;  it  is  especially  useful  for  the  demonstration 
of  spirochetes  in  secretions. 

Double  Microscopes  have  been  constructed  by  means  of  which 
a  comparative  study  may  be  made  of  two  objects  at  the  same  time. 

Microscopic  Examinations.  —  Bacteria  may  be  studied  micro- 
scopically, (1)  living  and  unstained  in  fluids,  (2)  in  stained  film 
preparations,  (3)  in  stained  sections  of  tissue.  For  such  studies 
perfectly  clean  slides  and  coverslips  are  necessary. 

Hanging  Drop  Preparation.  —  In  order  to  note  motility  or 
watch  the  method  and  rate  of  cell  division  it  is  necessary  to  study 
bacteria  while  they  are  alive.  This  can  be  accomplished  by 

means   of    the  so-called   hanging      

drop  prepared  as  follows :  A  spe- 
cial slide  with  a  circular  hollow 
on  one  surface  is  employed  and 
around  the  edge  of  the  concavity 
a  fine  film  of  cedar  oil  or  vaseline 


is  smeared.  This  latter  serves  the  FIG.  13.  —  Hanging  Drop  Prepara- 
purpose  of  attaching  the  coverslip 

to  the  slide  and  so  preventing  evaporation.  A  drop  of  fluid  in 
which  the  bacteria  are  growing  is  then  transferred  to  the  center 
of  a  coverslip  by  means  of  a  platinum  loop  previously  sterilized 
by  flaming.  If  the  bacteria  are  to  be  removed  from  solid  media 
or  are  obtained  from  thick  pus  they  are  mixed  with  a  suitable 
quantity  of  sterile  broth  or  physiological  salt  solution  (0.9  per 
cent  NaCl  in  distilled  water)  and  a  drop  of  the  suspension  placed 
on  the  coverslip,  or  the  bacteria  may  be  emulsified  in  a  drop  of 
salt  solution  directly  on  the  coverslip.  The  coverslip  is  then 
inverted  over  the  slide,  gently  pressed,  and  sealed  by  means  of  the 
vaseline  (Fig.  13). 

Unstained  organisms  are  difficult  to  see  through  the  microscope, 
therefore  great  care  is  necessary  in  focusing.  The  diaphragm 
should  be  partially  closed  in  order  to  take  advantage  of  the  lights 
and  shadows  caused  by  the  difference  in  light  transmission  in  the 
objects  under  examination.  Since  the  edge  of  the  hanging  drop 


42  BACTERIOLOGY  FOR  NURSES 

is  more  easily  distinguished  than  the  middle  it  should  be  found 
first  with  the  low-power  lens  and  then  so  arranged  that  the  edge 
of  the  drop  crosses  the  center  of  the  field.  The  high-power  objec- 
tive is  then  turned  into  position,  the  tube  lowered  until  it  almost 
touches  the  coverslip,  and  then  with  the  eye  at  the  ocular  it  should 
be  carefully  raised  into  focus. 

Hanging  Block.  —  The  manner  and  rate  of  cell  division  can 
perhaps  be  best  studied  by  means  of  the  hanging  block.  The 
technique  is  as  follows  :  A  thin  layer  of  melted  agar  is  poured  into 
a  Petri  dish  and  allowed  to  harden,  after  which  a  small  square 
is  removed,  seeded  with  the  organisms  to  be  studied  and  carefully 
placed  on  a  coverslip,  the  bacteria  being  between  the  agar  and  the 
glass.  The  preparation  is  placed  over  a  hollow  slide  and  examined 
under  the  microscope  in  the  manner  described  for  the  hanging  drop. 

Film  Preparation.  —  This  method  is  perhaps  the  most  frequently 
employed  of  all  for  bacterial  examination;  by  it  advantage  may 
be  taken  of  the  elective  properties  of  certain  bacteria  for  particular 
stains  and  a  diagnosis  be  thus  more  easily  established.  The 
steps  in  the  preparation  are  as  follows :  A  thin  even  film  of  the 
material  to  be  examined  is  spread  over  the  surface  of  a  perfectly 
clean  slide ;  if  the  material  is  fluid  it  is  transferred  by  means  of 
a  platinum  loop,. if  taken  from  solid  culture  medium  a  loopful  of 
sterile  water  is  first  placed  upon  the  slide  and  a  small  particle 
of  a  growth  thoroughly  mixed  with  it  before  spreading.  The 
film  is  allowed  to  dry  in  the  air.  The  slide  is  then  held  by  means 
of  a  pair  of  forceps  and  passed  slowly  through  the  flame  of  a  Bun- 
sen  burner  three  or  four  times,  film  side  uppermost,  in  order  to 
fix  the  preparation  on  to  the  slide.  Fixing  may  also  be  accom- 
plished after  the  film  is  dry  by  immersing  in  chemicals  such  as 
alcohol,  formalin,  glacial  acetic  acid,  etc.,  in  which  case  the  slide 
must  be  immersed  in  water  to  remove  the  excess  chemical  before  the 
next  step.  A  few  drops  of  the  desired  stain  are  then  placed  on 
the  slide  and  left  there  from  a  few  seconds  to  several  minutes 
according  to  the  dye  used.  The  slide  is  then  washed  carefully 
but  thoroughly  in  water,  after  which  it  is  dried  by  gently  pressing 
between  layers  of  filter  paper. 


EXAMINATION  AND  STAINING  OF  BACTERIA    43 

The  preparation  may  have  a  drop  of  cedar  oil  placed  directly 
upon  it  and  so  be  examined  under  the  oil  immersion  lens ;  if,  how- 
ever, the  slide  is  to  be  preserved  for  future  examinations  it  is  better 
to  first  place  a  small  drop  of  Canada  balsam  on  the  film  and  cover 
it  with  a  coverslip. 

Blood  Films.  —  A  blood  film  is  prepared  by  placing  a  drop  of 
blood  near  the  end  of  a  clean  slide  and  the  edge  of  a  second  slide 
is  then  lowered  on  to  the  drop 
at  an  angle  to  the  first  slide. 
By  capillarity  the  blood  spreads 
itself  along  the  edge  of  the 

Second     Slide,    which    is     gently        FIG.  14—Meth^of  making  Blood 

stroked  over  the  surface  of  the 

first,  leaving  a  film  the  width  and  thickness  of  which  depends  on 

the  angle  at  which  the  slide  was  held  (Fig.  14). 

Another  method  employed  is  to  place  a  drop  of  blood  between 
two  coverslips  and  then  draw  them  apart.  If  the  red  blood 
corpuscles  are  to  be  examined,  fixing  should  not  be  effected  by  heat ; 
the  slide  may  be  placed  in  a  mixture  of  equal  parts  of  alcohol  and 
ether  for  half  an  hour  and  then  washed  and  dried  in  the  air,  or  it 
may  be  placed  in  a  saturated  solution  of  corrosive  sublimate  for 
two  or  three  minutes  then  washed  well  in  running  water  and  dried. 
The  method  of  staining  depends  upon  the  object  for  which  the 
examination  is  to  be  made. 


THE   STAINING   OF  BACTERIA 

Staining  Principles.  —  Generally  speaking,  bacteria  react  to 
stains  in  a  manner  similar  to  the  nuclei  of  animal  cells ;  the  pro- 
cess is  to  be  regarded  as  a  chemical  combination  between  the  dye 
and  the  cell  substance  rather  than  merely  a  mechanical  saturation. 
Certain  organisms  stain  readily,  others  only  with  great  difficulty ; 
the  easily  stained  forms  require  immersing  but  a  few  minutes  in 
a  watery  dye  while  those  which  do  not  stain  so  readily  require  a 
longer  time  and  in  some  cases  the  addition  of  heat  or  a  mordant. 
The  tubercle  bacillus  belongs  to  the  latter  class.  Spores  and 


44  BACTERIOLOGY  FOR  NURSES 

flagella  are  also  stained  with  difficulty.  Those  organisms  that  do 
not  stain  readily  as  a  rule  retain  the  dye  and  are  not  easily  de- 
colorized. Two  explanations  are  advanced  for  this  resistance  to 
take  on  and  to  part  with  stains:  one  hypothesis  is  that  such 
organisms,  or  parts,  are  of  different  chemical  composition;  this 
assumption  is.  probably  true  in  the  case  of  spores  and  flagella. 
The  other  theory  supposes  the  presence  of  a  waxy  and  therefore 
difficultly  permeable  envelope  surrounding  the  bacteria  may  be 
the  cause.  The  latter  view  is  probably  correct  in  the  case  of  the 
tubercle  bacilli.  The  presence  of  a  waxy  or  fatty  material  has 
been  demonstrated  in  certain  bacteria ;  moreover,  when  this  waxy 
substance  has  been  extracted  with  ether  the  dye-resistant  qualities 
of  the  bacteria  have  disappeared  also.  It  may  be  in  certain  cases 
that  both  factors  combine  to  produce  the  result. 

The  best  bacterial  stains  are  derived  from  the  coal-tar  product 
aniline.  Many  of  the  dyes  have  the  constitution  of  salts  and  are 
divided  into  two  groups  according  as  the  staining  depends  on  the 
basic  or  acid  part  of  the  molecule.  The  basic  stains  have  a  special 
affinity  for  nuclear  material  and  the  acid  for  cytoplasm.  For 
this  reason  the  basic  dyes  are  especially  bacterial  stains. 

The  most  frequently  used  stains  are : 

Violet  —  Methyl  violet,  gentian  violet,  crystal  violet 
Blue  —  Methylene  blue,  thionin  blue 
Red  —  Basic  fuchsin 
Brown  —  Bismarck  brown 

The  violet  dyes  have  the  most  intense  action,  consequently 
care  should  be  taken  when  using  them  not  to  overstain  the  speci- 
men. Methylene  blue  is  perhaps  the  most  generally  employed ; 
it  gives  a  good  differentiation  of  structure  and  it  is  not  easy  to 
overstain  with  it.  Bismarck  brown  and  eosin  are  weak  dyes  and 
are  used  generally  as  counterstains. 

Saturated  Solutions.  —  It  is  a  convenient  arrangement  to  keep 
in  stock  saturated  solutions  of  the  dyes  most  frequently  employed 
and  from  them  to  make  dilutions  for  use  as  required.  Since  great 
variations  occur  between  different  samples  of  dyes  bearing  the 
same  name  no  definite  amount  can  be  quoted  as  the  minimum  for 


EXAMINATION   AND  STAINING  OF  BACTERIA    45 

the  preparation  of  a  saturated  solution.  A  complete  saturation 
may  be  obtained  by  adding  the  powdered  dye  to  the  solvent  until 
no  more  will  enter  into  solution,  a  slight  residue  remaining  after 
repeated  shaking  on  several  days  being  taken  as  an  indication. 
The  following  quantities  of  the  most  frequently  used  dyes  are 
approximately  sufficient  for  saturation : 

Gentian  violet :    1  to  5  per  cent  in  distilled  water  or  4  to  8  per  cent 

in  96  per  cent  alcohol 
Fuchsin :  1  to  5  per  cent  in  distilled  water  or  3  per  cent 

in  96  per  cent  alcohol 
Methylene  blue :  6  to  7  per  cent  in  distilled  water  or  7  per  cent 

in  96  per  cent  alcohol 

Stains  should  always  be  filtered  through  paper  before  use,  other- 
wise sediment  may  be  deposited  on  the  slide  which  would  spoil 
the  preparation. 

Mordants  and  Decolorizing  Agents.  —  Certain  organisms  are 
stained  with  difficulty  unless  a  mordant  is  employed,  which  not 
only  increases  the  intensity  of  the  dye  but  tends  to  make  the  bac- 
terial cell  more  permeable.  Again  in  films  of  blood  or  pus  and 
more  especially  in  sections  of  tissue,  the  tissue  cells  may  be  so 
deeply  stained  as  to  obscure  the  bacteria  lying  within  them.  To 
obviate  this  methods  have  been  devised  whereby  a  mordant  may 
be  used  to  fix  the  dye  in  the  bacteria  while  subsequent  treatment 
with  a  decolorizing  agent  will  remove  the  dye  to  a  greater  or  less 
extent  from  the  tissue  cells. 

Staining  properties  may  be  increased  by : 

(a)  The  addition  of  weak  solutions  of  alkalies,  such  as  caustic 

potash  or  ammonium  carbonate. 
(6)  The  addition  of  carbolic  acid,  aniline  oil,  metallic  salts, 

etc. 

(c)  Heat. 

(d)  Prolonged  staining. 

The  decolorizing  agents  generally  employed  are  weak  solutions  of 
acids,  alcohol,  or  a  combination  of  both. 


46  BACTERIOLOGY  FOR  NURSES 

FORMULA  OF  SOME  OF  THE  MOST  FREQUENTLY  USED  STAINS 
Loeffler  Methylene  Blue. 

Saturated  alcoholic  solution  of  methylene  blue  ...         30  c.c. 
1-10,000  solution  of  caustic  potash  in  distilled  water  .       100  c.c. 

Films  may  be  stained  with  the  above  preparation  for  from  two  to 
five  minutes  without  being  overstained.     It  is  a  useful  stain  for 
structural  differentiation  and  is  generally  employed  in  routine 
examination  for  the  diphtheria  bacillus. 
Neisser's  Stain.  —  Two  solutions  are  used : 

(a)  5  per  cent  alcoholic  solution  of  methylene  blue     .  20  c.c. 

Glacial  acetic  acid 50  c.c. 

Distilled  water 950  c.c. 

(6)  Bismarck  brown 2  gm. 

Distilled  water 1000  c.c. 

Films  are  stained  in  (a)  for  three  to  five  seconds,  washed  in  water 
and  stained  in  (6)  for  five  seconds,  dried  and  examined. 

The  stain  was  originally  introduced  by  Neisser  as  an  aid  in  the 
identification  of  the  diphtheria  bacillus.  If  the  film  is  made 
from  a  twenty-four  hour  culture  grown  on  serum  medium  the 
bacilli  frequently  show  when  stained  by  this  method  deep  blue 
granules  with  surrounding  protoplasm  of  a  faint  brown. 

Ziehl-Neelsen's  Carbol  Fuchsin  Stain. 

Basic  fuchsin 1  gm. 

Absolute  alcohol 10  c.c. 

5  per  cent  solution  of  carbolic  acid 100  c.c. 

After  fixing  the  film  may  be  placed  in  the  stain,  heated  until  steam 
rises  and  allowed  to  remain  there  for  five  minutes,  or  it  may  be 
allowed  to  remain  in  the  cold  stain  from  twelve  to  twenty-four 
hours.  The  excess  of  stain  is  then  washed  off  with  water  and  the 
film  placed  in  a  decolorizing  solution ;  3  per  cent  hydrochloric  acid 
in  80  per  cent  alcohol  gives  good  results  as  a  decolorizing  agent. 
After  a  few  seconds  remove  the  film  and  wash  in  water ;  if  only  the 
faintest  pink  color  persist  decolorization  has  been  sufficient;  if 


EXAMINATION  AND   STAINING   OF  BACTERIA    47 

on  the  other  hand  a  distinctly  red  color  remains  the  process  should 
be  repeated  until  the  proper  tint  is  obtained.  The  slide  is  next 
immersed  in  a  10  per  cent  watery  solution  of  methylene  blue, 
washed  in  water,  and  dried. 

The  above  method  is  used  for  the  group  of  organisms  known 
as  "  acid-fast,"  amongst  which  are  the  tubercle  bacillus,  the 
leprosy  bacillus,  the  smegma  bacillus,  the  hay  bacillus,  and  a 
number  of  others.  These  organisms  require  a  powerful  dye  con- 
taining a  mordant,  and  the  staining  process  must  be  continued 
a  long  time  or  its  action  aided  by  the  application  of  heat.  When 
once  stained,  however,  they  resist  the  decolorizing  action  of 
strong  acids ;  for  this  reason  they  are  spoken  of  as  "  acid-fast." 
Stained  by  the  above  method  they  appear  under  the  microscope 
to  be  bright  red  while  any  other  organisms  present  take  the 
counterstain  and  appear  blue. 

Spore  Stain.  Moeller's  Method.  —  The  films  are  immersed 
in  chloroform  for  two  minutes,  washed  in  water,  then  covered  with 
5  per  cent  chromic  acid  one  minute  and  again  washed  in  water. 
They  are  next  placed  in  carbol  fuchsin  and  the  solution  is  heated 
until  it  commences  to  steam.  After  remaining  in  the  hot  solution 
for  three  minutes  the  slides  are  removed,  washed  in  water,  and 
decolorized  in  5  per  cent  sulphuric  acid  for  five  to  ten  seconds, 
then  washed  in  water  and  counterstained  in  10  per  cent  aqueous 
methylene  blue  one  minute.  By  this  method  the  spores  appear  red 
while  the  remainder  of  the  bacterial  cell  is  stained  blue.  If  bacilli 
containing  spores  are  stained  with  a  watery  solution  of  any  of 
the  aniline  dyes  the  spores  remain  unstained  and  appear  as  clear 
spaces  surrounded  by  stained  protoplasms. 

Capsule  Stain.  Hiss'  Method.  —  Films  are  made  in  the  usual 
way  and  fixed  by  heat.  The  slide  is  then  covered  with  a  5  per 
cent  watery  solution  of  fuchsin  or  gentian  violet  and  heated  over 
a  Bunsen  flame  until  it  steams.  The  dye  is  washed  off  with  a 
20  per  cent  aqueous  copper  sulphate  solution,  after  which  it  is 
dried  between  layers  of  filter  paper  without  further  washing  in 
water.  By  this  method  the  capsule  appears  as  a  faint  blue  halo 
surrounding  a  dark  purple  or  red  cell  body. 


48  BACTERIOLOGY  FOR  NURSES 

Flagella  Stain.  Van  Ermengen's  Method.  —  Three  solutions 
are  necessary : 

(1)  Twenty  per  cent  tannic  acid  solution      ....    60  c.c. 

Two  per  cent  osmic  acid  solution 30  c.c. 

Glacial  acetic  acid 4  to  5  drops 

The  fixed  film  is  placed  in  this  solution  for  one  hour  at  room 
temperature  or  for  five  minutes  at  100°  C.  It  is  then  washed 
in  water  and  afterwards  in  absolute  alcohol,  followed  by  immersion 
for  one  to  three  seconds  in 

(2)  Silver  nitrate  3  to  5  per  cent  solution.  Without  washing  the  slide 
is  transferred  to 

(3)  Gallic  acid 5  gm. 

Tannic  acid 3  gm. 

Fused  potassium  acetate  .........  10  gm. 

Distilled  water 350  c.c. 

The  slide  should  be  moved  gently  to  and  fro  in  this  solution  for  a 
few  minutes,  then  returned  to  the  silver  nitrate  until  the  film  turns 
black.  It  is  then  thoroughly  washed  in  water  and  dried. 

The  staining  of  flagella  is  one  of  the  most  difficult  of  bacteriologi- 
cal procedures.  In  order  to  get  good  results  the  slide  must  be 
scrupulously  clean,  the  film  should  be  made  from  a  young  ten  to 
eighteen  hour  agar  culture  and  should  be  spread  as  carefully  and 
with  as  little  manipulation  as  possible. 

Indian  Ink  Method  for  the  Examination  of  Spirochetes.  - 
An  emulsion  of  good  quality  Indian  ink  is  sterilized  by  steaming 
and  allowed  to  settle  for  a  few  days.  One  drop  of  the  sediment 
and  one  drop  of  water  are  thoroughly  mixed  with  a  loopful  of  the 
material  to  be  examined  on  a  clean  slide.  The  film  is  dried  in  the 
air  and  examined  with  the  oil  immersion  lens.  If  spirochetes 
are  present  they  stand  out  unstained  surrounded  by  the  dark 
Indian  ink. 

Wright's  Stain.  —  This  is  one  of  several  modifications  of  the 
polychrome  Romanowsky  stains  used  chiefly  for  staining  animal 
cells  and  also  for  bacteria  that  stain  faintly  by  ordinary  methods. 
It  can  be  purchased  ready  for  use  or  it  may  be  prepared  as  follows : 
1  per  cent  methylene  blue  and  0.5  per  cent  sodium  carbonate  are 


EXAMINATION  AND  STAINING   OF  BACTERIA    49 

mixed  and  steamed  in  the  sterilizer  for  one  hour.  When  cold  add 
0.1  per  cent  aqueous  solution  of  eosin  in  the  proportion  of  5  parts 
eosin  solution  to  6  parts  methylene  blue  solution.  The  mixture 
becomes  a  purple  color  and  a  granular  sediment  appears.  It  is 
then  filtered  and  the  precipitate  remaining  on  the  filter  paper  is 
pressed  dry.  A  saturated  solution  is  made  of  the  dried  precipitate 
in  methyl  alcohol;  this  saturated  solution  is  then  filtered  and 
diluted  by  the  addition  of  10  c.c.  of  methyl  alcohol  to  40  c.c.  of 
the  stain. 

In  using  the  stain  a  few  drops  are  placed  on  a  fixed  film  for 
one  minute,  then  the  same  quantity  of  water  is  dropped  on  to 
the  slide  by  means  of  a  medicine  dropper  and  the  mixture  of  stain 
and  water  is  allowed  to  remain  two  to  three  minutes.  The  slide 
is  then  washed  in  water  and  dried. 

The  stain  gives  particularly  good  results  in  the  examination 
of  blood  films.  Erythrocytes  appear  yellow  or  pink;  the  nuclei 
of  leucocytes  various  shades  of  purple  and  the  cytoplasm  a  light 
blue  color;  blood  plaques  dark  blue;  bacteria  blue.  Malarial 
parasites  stain  characteristically :  the  chromatin  mass  appears 
a  garnet  red  and  the  surrounding  protoplasm  a  robin's  egg  blue. 

Gram's  Stain.  —  The  fixed  film  is  covered  with  a  fresh  solution 
of  aniline  gentian  violet  made  as  follows :  One  c.c.  of  anilin  oil  is 
added  to  10  c.c.  of  water  and  shaken  until  thoroughly  emulsified, 
after  which  it  is  filtered  through  wet  filter  paper.  One  part  of 
saturated  alcoholic  gentian  violet  is  then  added  to  nine  parts  of 
the  filtrate.  The  slide  is  allowed  to  remain  in  the  above  dye  for 
five  minutes,  after  which  it  is  immersed  in  the  following  iodine 
solution  for  2  to  3  minutes. 

Iodine 1  gm. 

Potassium  iodide 2  gm. 

Distilled  water 300  c.c. 

The  film  is  then  decolorized  with  97  per  cent  alcohol  about  one 
minute  or  until  no  more  stain  can  be  washed  out  of  the  preparation, 
after  which  it  is  washed  in  water  and  counterstained  with  eosin 
for  thirty  seconds. 
This  method  of  staining  is  frequently  used  in  bacterial  differen- 


50  BACTERIOLOGY   FOR  NURSES 

tiation.  By  its  means  organisms  are  divided  into  two  classes : 
those  which  retain  the  initial  stain  are  spoken  of  as  Gram  positive 
and  those  which  are  decolorized  and  take  the  counterstain  as  Gram 
negative.  Most  bacteria  fall  decidedly  into  one  class  or  the  other ; 
borderline  cases  do  occur,  however,  and  a  few  species  show  a  tend- 
ency to  change  from  Gram  positive  to  Gram  negative  in  old  cul- 
tures. 

CLASSIFICATION  OF  THE  PRINCIPAL  PATHOGENIC  BACTERIA  ACCORDING 
TO  THEIR  REACTION  TO  GRAM'S  STAIN 

POSITIVE  NEGATIVE 

(Retain  the  violet  stain)  (Take  the  counterstain) 

Cocci  Cocci 

M.  tetragenus  M.  catarrhalis 

Pneumococcus  group  M.  gonorrheas 

Staphylococcus  group  M.  meningitidis 

Streptococcus  group  M.  melitensis 

Bacilli  Bacilli 

B.  serogenes  capsulatus  B.  acidi  lacti 

B.  anthracis  B.  coli  group 

B.  botulinus  B.  dysenterise  group 

B.  diphtherise  group  B.  enteritidis  group 

B.  tetani  B.  influenzas  group 

B.  tuberculosis  and  other  acid-  B.  Koch- Weeks 

fast  bacilli  B.  lactis  aerogenes 

B.  maligni  edematis 

B.  mallei 

B.  Morax  Axenfeld 

B.  mucosus  capsulatus 

B.  pertussis  group 

B.  pestis 

B.  proteus   . 

B.  pyocyaneus 

B.  typhosus  group 

Spirillum 
S.  choleras 


CHAPTER  V 

CULTIVATION  AND    IDENTIFICATION  OF    BACTERIA 

General  Laboratory  Rules.  —  A  jar  containing  1  in  20  carbolic 
acid  solution  should  be  always  at  hand  in  which  to  place  all  glass- 
ware that  has  been  used  for  infective  material;  after  several 
hours  such  articles  may  be  cleansed  by  boiling  in  soapsuds.  Old 
used  cultures  may  be  sterilized  in  the  Arnold  for  three  or  four  hours 
or  for  a  shorter  period  in  the  autoclave.  It  is  well  to  have  within 
easy  reach  a  basin  of  mercuric  chloride  1  in  1000  or  carbolic  acid 
1  in  40  in  which  the  worker's  hands  may  be  disinfected  in  case  of 
accidental  contamination.  Any  infective  material  spilled  on  the 
table  should  be  covered  with  carbolic  solution  and  carefully  wiped 
off  with  cotton  held  by  forceps.  Unnecessary  movement  in  the 
laboratory  should  be  avoided  in  order  that  the  air  may  be  kept  as 
quiet  as  possible.  Hands  should  always  be 
well  washed  before  leaving  the  laboratory  and 
food  should  not  be  eaten  there.  Labels  should 
never  be  moistened  with  the  tongue  and 
nothing  should  be  placed  in  the  mouth  that 
has  touched  any  surface  in  the  laboratory. 

Cultivation  of  Microorganisms.  —  In  order 
to  learn  the  special  characteristics  of  an  or- 
ganism it  must  be  studied  apart  from  all 
other  forms  on  artificial  culture  media  such  as 
already  described.  A  large  surface  for  growth 
is  obtained  by  filling  tubes  with  solid  media 
such  as  agar  or  gelatin  about  one  sixth  full,  and 
after  sterilization,  while  still  liquid,  placing 
them  in  a  slanting  position  so  that  when  solidified  they  will  give  an 
oblique  surface  of  three  to  four  inches  (Fig.  15).  Care  should  be 
taken  that  the  medium  does  not  extend  to  the  cotton  plug. 

51 


FIG 


15.—  A.  Tubed 
Agar  for  Stab  Cul- 
ture. B.  Agar  Slant. 


52  BACTERIOLOGY   FOR  NURSES 

A  tube  of  media  in  which  bacteria  are  growing  is  spoken  of  as 
a  "  culture."  When  only  one  species  is  present  it  is  said  to  be  a 
"  pure  culture."  If  a  small  portion  of  an  already  existing  growth 
is  transferred  to  a  tube  of  fresh  medium  the  resulting  culture  is 
spoken  of  as  a  "  transplant  "  or  "  subculture."  To  transfer 
bacteria  from  one  tube  to  another  a  platinum  wire  or  loop  is  used. 

It  should  be  thin 
yet  sufficiently  stiff 
and  about  two  and 
a  half  inches  long. 

FIG.  16.  —  Platinum  Wire  and  Loop.  r  jg      ftt_ 


tached  to  an  aluminum  holder  or  fused  into  the  end  of  a  glass  rod 
about  seven  or  eight  inches  long.  For  making  stab  cultures  or 
for  "  fishing  "  a  straight  "  needle  "  or  wire  is  used  ;  for  transfer- 
ring growth  from  a  fluid  medium  a  wire  turned  at  the  end  to  form 
a  loop  is  employed  (Fig.  16).  The  platinum  needle  or  loop  should 
always  be  sterilized  in  the  flame  of  a  Bunsen  burner  immediately 
before  and  after  using. 

A  subculture  is  made  as  follows  :  The  culture  and  the  tube 
to  be  inoculated  are  held  in  a  slanting  position  between  the  thumb 
and  the  first  and  second  fingers  of  the  left  hand  ;  the  plugs  are 
gently  twisted  around  once  or  twice 
to  make  sure  they  are  not  sticking 
to  the  tubes  and  if  they  have  been 
exposed  to  dust  they  should  be 
slightly  singed  in  the  gas  flame. 
The  platinum  wire,  held  between 
the  thumb  and  the  first  and  second 

......  .  FIG.  17.  —  Method  of  Inoculating. 

fingers  ot  the  right  hand,  somewhat 

in  the  manner  of  holding  a  pen,  is  sterilized  by  heating  red  hot  in 
a  flame.  The  plugs  are  then  removed,  one  is  grasped  between 
the  middle  and  third  fingers  and  the  other  between  the  third  and 
fourth  fingers.  When  cool  enough  the  wire  is  carefully  passed  into 
the  culture  tube  and  without  touching  the  side  a  small  amount  of 
bacterial  growth  is  removed  and  immediately  transferred  to  the 
tube  which  is  to  be  inoculated  (Fig.  17).  If  the  tube  contains 


CULTIVATION  OF   BACTERIA  53 

slanted  medium  the  growth  is  deposited  by  lightly  smearing  the 
surface  from  the  lower  to  the  upper  portion  of  the  slant.  A 
"  stab  "  culture  is  made  by  plunging  the  needle  down  the  center 
of  medium  that  has  not  been  slanted  or  the  two  methods  may  be 
combined  on  an  agar  slant;  growth  may  be  smeared  on  the 
surface  and  the  needle  plunged  into  the  medium  before  it  is 
withdrawn.  Surface  growth  and  deep  growth  may  thus  be  ob- 
served in  the  same  tube.  If  the  organisms  are  transplanted 
from  one  fluid  medium  to  another  a  loopful  is  removed  and  gently 
rubbed  off  against  the  glass  in  the  upper  portion  of  the  fresh 
medium.  When  the  growth  forms  a  pellicle  it  is  sometimes 
necessary  to  transfer  a  portion  of  the  pellicle  and  so  place  it 
that  it  rests  on  the  surface  of  the  fresh  medium,  or  growth  will 
not  take  place.  After  the  medium  is  inoculated  the  platinum 
needle  is  removed,  the  plugs  replaced,  and  the  wire  immediately 
heated  red  hot  in  the  flame ;  at  the  same  time  several  inches  of  the 
glass  rod  should  be  passed  through  the  flame  also. 

Plating.  —  If  pathological  material  or  such  substances  as  milk 
or  water  be  placed  in  culture  medium  many  different  kinds  of 
organisms  develop  at  the  same  time.  Since  it  is  impossible  to 
study  the  characteristics  of  each  species  unless  they  can  be  sepa- 
rated, a  method  devised  by  Koch  of  plating  in  solid  media  is  em- 
ployed whereby  individual  bacteria  are  held  apart  and  the  descend- 
ants of  each  are  soon  sufficiently  numerous  to  appear  as  a  colony 
visible  to  the  naked  eye.  By  a  procedure  known  as  colony  fishing 
the  members  of  a  single  species  can  be  transferred  to  fresh  sterile 
media  and  a  pure  culture  thus  obtained. 

A  thin  layer  of  medium  presenting  a  moderately  large  surface 
is  obtained  by  the  use  of  the  so-called  Petri  dish.  Each  Petri 
dish  consists  of  two  circular  glass  plates,  the  larger  forming  a  loosely 
fitting  lid  for  the  smaller. 

The  method  of  making  a  pour  plate  for  the  isolation  of  bacteria 
is  as  follows :  The  solid  medium  is  liquefied  and  then  cooled  to  a 
temperature  of  42°  C.  A  loopful  of  the  material  to  be  examined 
is  placed  in  a  tube  and  thoroughly  mixed  with  the  medium  by 
rolling  the  tube  between  the  hands,  care  being  taken  not  to  shake 


64  BACTERIOLOGY  FOR  NURSES 

the  tube  and  produce  air  bubbles.  Three  loopfuls  of  this  mixture 
are  transferred  to  a  second  tube  and  the  mixing  process  repeated ; 
five  loopfuls  from  the  second  tube  are  carried  to  a  third  and  mixed. 
The  necks  of  the  tubes  are  then  flamed  and  the  contents  of  each 
poured  into  a  Petri  dish  previously  marked  with  the  number  one, 
two,  or  three  according  to  the  dilution  it  is  to  receive.  In  pouring 
the  inoculated  medium  into  the  Petri  dish  the  cover  should  be 
raised  along  one  margin  just  high  enough  to  permit  the  entrance 

of  the  neck  of  the  tube,  care 
being  taken  that  the  sides  of 
the  tube  do  not  touch  either 
the  top  or  bottom  part  of  the 
dish  (Fig.  18).  The  cover  is 
immediately  replaced  and  the 

FIG.  18.  —  Method  of  pouring  Media        ,.  ,  .,  ,     ,  .. 

into  a  Petri  Dish.  dish  gently  rotated  if  necessary 

to  insure  the  even  distribution 
of  the  medium  over  the  bottom  of  the  dish  before  it  solidifies. 

It  is  advisable  to  make  a  stained  film  preparation  of  the  material 
to  be  examined  before  plating  as  above,  in  order  to  have  some  idea 
of  the  number  of  organisms  present.  If  they  are  relatively  few, 
more  material  should  be  inoculated  into  the  first  tube ;  if  numerous, 
then  a  further  dilution  will  be  necessary. 

When  agar  has  been  used  for  plating,  the  Petri  dishes  are  inverted 
as  soon  as  the  medium  has  solidified  and  placed  in  the  incubator 
at  a  temperature  of  37°  C. ;  gelatin  plates  are  kept  in  a  dark  place 
at  room  temperature.  Colonies  will  develop  in  from  one  to  three 
days  according  to  the  species,  and  in  the  second  or  third  dilution 
they  will  usually  be  found  sufficiently  far  apart  to  be  "  fished." 

Surface  Streaking.  —  When  it  is  desired  to  isolate  bacteria 
which  are  particularly  sensitive  to  their  surroundings,  such  as  the 
gonococcus,  and  which  require  special  medium,  surface  streaking 
instead  of  the  dilution  method  is  employed.  The  technique  is  as 
follows  :  Pour  plates  are  made  of  suitable  medium  and  when  hard- 
ened the  material  containing  the  organisms  to  be  isolated  is  smeared 
over  the  surface  of  several  plates  in  succession.  If  the  organisms 
are  removed  from  the  nose  or  throat  by  means  of  a  swab,  the  swab 


CULTIVATION  OF  BACTERIA  55 

is  gently  stroked  over  the  medium  in  plate  one,  and  then,  without 
turning,  the  same  portion  of  the  swab  is  smeared  over  a  second  and 
a  third  plate.  If  the  material  to  be  examined  is  liquid  a  small 
quantity  is  deposited  on  .the  surface  of  the  medium  by  means  of  a 
platinum  loop  and  the  loop  is  then  stroked  lightly  over  the  medium 
in  several  plates  without  recharging. 

A  method  of  plating  is  frequently  used  by  means  of  which  the 
dilutions  are  carried  out  in  definite  proportions  so  that  the  num- 
ber of  bacteria  present  may  be  estimated. 

The  procedure  is  as  follows :  To  9  c.c.  of  sterile  water  1  c.c. 
of  the  material  to  be  examined  is  added  and  the  resulting  mixture 
is  thoroughly  shaken  to  separate  the  organisms.  One  c.c.  of  this 
1  in  10  dilution  is  added  to  9  c.c.  of  sterile  water,  producing  a  1 
in  100  dilution.  After  thorough  shaking  the  process  may  be 
repeated,  giving  as  a  result  a  1  in  1000  dilution,  and  so  on  until 
the  desired  limit  has  been  reached.  One  c.c.  of  each  dilution  is 
placed  by  means  of  a  sterile  pipette  in  a  Petri  dish  previously 
numbered,  and  melted  agar  cooled  to  40°  C.  is  poured  into  the 
center  of  it.  Mixing  is  accomplished  by  gently  rotating  the  plates 
before  the  medium  solidifies,  care  being  taken  that  the  medium 
remains  flat  on  the  bottom  and  is  not  smeared  over  the  sides. 
After  growth  has  occurred  the  colonies  are  counted  and  the  number 
of  bacteria  present  in  the  material  examined  estimated. 

For  example,  if  on  the  Petri  dish  containing  the  1  in  100  dilu- 
tion 180  colonies  appear  that  number  is  multiplied  by  100,  and  it 
is  assumed  that  approximately  18,000  organisms  were  present 
in  1  c.c.  of  the  material  examined.  The  dilution  showing  between 
one  hundred  and  two  hundred  colonies  is  chosen  as  the  most 
representative  for  counting.  Higher  numbers  are  difficult  to  count ; 
also  crowding  may  have  checked  the  development  of  some  of  the 
organisms.  Counting  may  be  facilitated  by  dividing  the  Petri 
dish  into  sections  by  lines  made  with  a  colored  pencil  or  by  placing 
the  dish  on  a  Wolffhiigel  counting  plate  (Fig.  19).  Whenever 
possible  all  the  colonies  should  be  counted ;  if  they  are  too  numer- 
ous and  a  counting  plate  is  used,  squares  from  representative  parts 
of  the  dish  are  counted  and  the  total  number  of  colonies  estimated. 


56  BACTERIOLOGY  FOR  NURSES 

As  sixty-three  squares  would  cover  the  standard  Petri  dish  the 
number  of  colonies  in  one  square  multiplied  by  sixty-three  would 
give  the  total  number  present  if  they  were  evenly  distributed 
throughout  the  medium.  Since  this  rarely  occurs  it  is  best  to 
count  ten  representative  squares  and  multiply  the  resulting  num- 


FIG.  19.  —  Wolffhiigel  Counting  Plate. 

ber  by  6.3.  This  is  in  turn  multiplied  by  the  dilution  used  to  give 
the  number  of  bacteria  present  in  the  material  examined.  Briefly 
it  may  be  expressed  thus : 

Number  of  colonies  in  10  squares  X  6.3  X  dilution 

=  Colonies  developed  from  1  c.c.  of  material  plated. 

The  above  method  is  very  satisfactory  when  only  one  species  of 
bacteria  is  present  and  the  medium  and  surrounding  conditions 
are  known  to  be  suitable  to  their  growth.  If  on  the  other  hand 
the  material  contains  a  number  of  different  species,  it  is  not  likely 
that  the  optimum  conditions  for  one  will  be  the  optimum  condi- 
tions for  all,  and  many  organisms  may  not  develop  into  colonies 


CULTIVATION   OF  BACTERIA  57 

at  all.  In  the  latter  case  the  method  does  not  give  absolutely 
accurate  information,  nevertheless  it  is  the  best  known  for  the 
routine  examination  of  water  and  milk. 

Fishing.  —  When  the  object  of  plating  is  to  obtain  a  pure  cul- 
ture rather  than  the  enumeration  of  the  organisms  present,  a  colony 
is  fished  from  the  Petri  dish  to  a  tube  of  fresh  culture  medium. 
Fishing  is  best  accomplished  by  using  the  microscope ;  otherwise  a 
minute  colony  of  another  species  unseen  by  the  naked  eye  may  be 
touched  and  the  resulting  subculture  contaminated.  The  lower 
part  of  the  Petri  dish  is  placed  on  the  stage  of  the  microscope 
and  the  low-power  lens  focused  over  the  colony  chosen.  The 
sterilized  platinum  needle  is  held  in  the  right  hand  and  introduced 
between  the  objective  and  the  colony.  When  the  point  of  the 
needle  is  visible  through  the  microscope  it  is  gently  lowered  until 
it  is  seen  to  touch  the  colony  and  to  carry  away  a  small  portion 
of  it.  The  needle  is  then  withdrawn  without  being  permitted  to 
touch  anything  in  passing  and  the  organisms  clinging  to  it  are 
transferred  to  the  medium  desired.  Fishing  requires  practice ; 
only  by  careful  manipulation  can  the  subculture  be  obtained 
pure. 

Incubation.  —  The  temperature  at  which  the  inoculated  media 
should  be  kept  depends  largely  upon  the  organisms  to  be  grown. 
Many  saprophytes  are  accustomed  to  the  temperature  of  an  ordi- 
nary room  and  consequently  no  special  apparatus  is  necessary  for 
their  cultivation.  Pathogenic  organisms,  on  the  other  hand, 
require  the  body  temperature  37.5°  C.  for  their  best  growth. 

In  order  to  maintain  a  constant  temperature  at  any  required 
degree  an  incubator  is  employed.  Different  makes  vary  somewhat 
in  detail  but  all  are  constructed  on  much  the  same  principle.  Gen- 
erally speaking  an  incubator  is  a  double-walled  copper  chamber 
with  double  doors ;  the  space  within  the  two  walls  is  filled  with 
water,  which  being  a  poor  conductor  of  heat  prevents  rapid  changes 
of  temperature  taking  place  within  the  chamber  as  a  result  of 
changes  on  the  exterior.  It  may  be  heated  by  electricity,  gas,  or 
oil ;  a  thermo-regulator  is  usually  attached  to  automatically  con- 
trol the  flame. 


58  BACTERIOLOGY   FOR  NURSES 

Anaerobic  Methods.  —  Special  methods  have  been  devised  for 
the  cultivation  of  anaerobic  organisms,  the  principle  of  each  being 
the  removal  of  free  oxygen  from  the  container  in  which  the  bac- 
teria are  growing.  The  earliest  methods  depended  on  the  removal 
of  oxygen  by  mechanical  means;  later,  other  methods  were  em- 
ployed whereby  the  oxygen  might  be  displaced  by  an  inert  gas 
such  as  hydrogen  or  absorbed  by  an  alkaline  solution  of  pyrogallol. 

Mechanical  Exclusion  of  Air.  —  Tubes  of  glucose  agar  or  glu- 
cose gelatin  are  heated  and  then  rapidly  placed  in  ice  water  to  pre- 
vent reabsorption  of  oxygen  while  the  medium  is  hardening.  The 
tubes  are  inoculated  by  deep  stabs  after  which  the  surface  may  be 
covered  with  a  layer  of  albolene;  or  tight-fitting  corks  covered 
with  sealing  wax  or  paraffin  may  be  used  to  replace  the  cotton 
plugs. 

Displacement  of  Air  by  Hydrogen.  —  This  method  consists  in 
passing  a  stream  of  hydrogen  through  an  airtight  chamber  in  which 
the  tubes  or  plates  of  inoculated  media  have  been  placed.  Hydro- 
gen may  be  generated  from  a  mixture  of  zinc  and  sulphuric  acid 
in  a  Kipp's  generator  which  is  connected  with  a  Novy  jar  contain- 
ing the  cultures.  Hydrogen  is  allowed  to  flow  through  the  jar 
about  ten  minutes  and  the  stopcocks  are  then  closed. 

Chemical  Absorption  of  Oxygen.  —  Any  vessel  with  a  tight 
cover  such  as  a  Novy  jar  or  a  Mason  fruit  jar  may  be  employed. 
Dry  pyrogallic  acid  is  placed  in  the  bottom  of  the  jar  and  a  small 
quantity  of  5  per  cent  sodium  hydroxide  is  poured  over  it.  The 
inoculated  tubes  are  put  in  place  and  the  jar  immediately  closed. 
If  plated  cultures  are  being  cultivated  the  plates  must  be  raised 
above  the  pyrogallic  mixture.  The  method  may  be  applied  to 
individual  tubes ;  the  inoculated  tube  is  placed  in  a  larger  one  in 
the  bottom  of  which  pyrogallic  acid  and  sodium  hydroxide  solu- 
tion have  been  placed.  The  larger  tube  is  closed  with  a  tight-fitting 
rubber  stopper.  Still  another  method  is  to  place  the  pyrogallic 
mixture  in  a  tumbler  and  invert  the  inoculated  tube  of  solid  medium 
into  it.  A  layer  of  oil  is  then  run  over  the  surface  of  the  pyrogallic 
acid  to  prevent  the  access  of  atmospheric  oxygen.  As  the  acid 
absorbs  oxygen  it  becomes  first  pale  yellow,  finally  deepening  to 


IDENTIFICATION  OF  BACTERIA  59 

a  dark  brown.     When  the  color  ceases  to  darken  it  may  be  assumed 
that  all  the  surrounding  oxygen  has  been  absorbed. 


IDENTIFICATION   OF   SPECIES 

When  it  is  desired  to  identify  an  unknown  organism  already 
isolated  in  pure  culture  a  study  is  made  of  its  general  characteris- 
tics. 

A.  Morphology,  method  of  grouping,  motility,  spore  formation, 
and  staining  reactions. 

B.  Cultural  reactions. 

C.  Effect  on  animals. 

A.  Morphology  and  Staining  reaction  may  be  determined  by 
film  preparations  and  motility  by  means  of  a  hanging  drop  made 
from  a  twenty-four-hour  broth  culture.     To  test  for  spore  forma- 
tion a  film  may  be  made  from  a  forty-eight-hour   culture   and 
stained  by  the  method  already  described,  or  the  culture  may  be 
heated  to  75°  C.  for  half  an  hour,  after  which  a  subculture  should 
be  made  and  incubated.     No  vegetative  forms  will  be  found  to 
resist  that  temperature. 

B.  Cultural  Reactions.  —  Ordinarily,  young  cultures  of  twenty- 
four  hours'  growth  should  be  observed  and  the  following  points 
noted : 

(1)  Growth  on  agar  plates.  —  Size  of  colony,  outline,  transpar- 
ency, texture,  color.  (The  colonies  should  be  observed 
with  a  hand  lens  or  under  the  low-power  objective  of  the 
microscope.) 

;(2)  Surface  growth  on  agar  slant  at  37°  C.  and  22°  C.  —  Scanty 
or  abundant,  smooth  or  irregular,  moist  or  dry,  slimy 
or  brittle. 

(3)  Growth  in  stab  culture.  —  Most  abundant  at  the  top  or 

bottom,  extension  of  growth  into  medium  (an  indica- 
tion of  motility).     Growth  a  continuous  line  or  beaded. 

(4)  Growth  in  broth.  —  Cloudy  at  upper  or  lower  level  or 

throughout,  pellicle  or  sediment  formation. 


60  BACTERIOLOGY  FOR  NURSES 

(5)  Productive  pigment.  —  Potato   medium   is   best   for   this 

purpose ;  if  it  is  not  available  a  little  of  the  growth  may 
be  taken  from  an  agar  slant  with  a  platinum  loop  and 
spread  on  white  paper,  when  the  color  will,  if  present, 
stand  out  against  the  white  background. 

(6)  Food  requirements.  —  Growth  on  simple  media  or  necessity 

for  media  containing  sugar  or  body  fluids. 

(7)  Temperature.  —  Minimum  —  optimum  —  maximum. 

(8)  Oxygen  requirement.  —  Growth  only  in  the  presence  of 

free  oxygen  on  the  surface.  Growth  only  in  the  absence 
of  free  oxygen  at  the  bottom  of  stab  culture. 

(9)  Proteolytic  action.  —  Liquefaction  of  gelatin ;  indol  forma- 

tion. For  the  latter  test  peptone  water  medium  with- 
out the  addition  of  sugar  is  employed.  The  culture  is 
incubated  for  from  four  to  six  days,  after  which  1  c.c. 
of  a  10  per  cent  solution  of  sulphuric  acid  and  1  c.c.  of 
a  1  in  10,000  solution  of  sodium  sulphite  is  added.  At 
the  point  where  the  acid  comes  in  contact  with 
the  medium  a  pink  color  appears  in  the  presence  of 
indol. 

(10)  Fermentation  of  sugars.  —  The  tests  usually  employed  are 
for  the  detection  of  acid  and  gas  production.  The  dif- 
ferent species  of  bacteria  vary  greatly  in  their  ability 
to  break  down  the  various  sugars;  one  species  may 
have  the  power  to  produce  acid  from  one  kind  of 
sugar,  acid  and  gas  from  another,  and  yet  have  abso- 
lutely no  effect  upon  a  third.  The  medium  employed  is 
generally  peptone  solution  to  which  has  been  added 
1  per  cent  of  the  sugar  chosen  and  an  indicator  such 
as  litmus  or  neutral  red.  Hiss's  serum  water  is  fre- 
quently used  for  the  detection  of  acid ;  the  cleavage  of 
the  carbohydrate  is  indicated  not  only  by  a  changed 
color  of  the  indicator  but  also  by  the  coagulation  of 
the  serum. 

The  test  for  acid  and  gas  production  may  be  carried 
on  at  the  same  time  in  a  specially  devised  fermentation 


IDENTIFICATION  OF  BACTERIA  61 

tube  (Fig.  11).  The  tube  is  filled  beyond  the  bend  with 
the  colored  sugar  medium  and  sterilized  by  the  discon- 
tinuous method.  After  inoculation  it  is  incubated,  and 
if  the  organism  is  capable  of  producing  gas  from  the 
sugar  present  the  gas  will  be  found  to  have  accumulated 
in  the  closed  arm  and  the  medium  displaced  into  the 
bulb.  If  after  twenty-four  to  forty-eight  hours  the 
column  of  gas  no  longer  increases  the  tube  may  be 
removed  and  the  amount  noted.  The  gas  produced  is 
mainly  carbon  dioxide  and  hydrogen.  A  rough  esti- 
mate of  the  percentage  of  each  may  be  made  by  first 
marking  the  tube  at  the  line  of  displaced  medium,  then 
filling  the  bulb  with  a  solution  of  caustic  soda.  The 
cotton  plug  is  replaced  by  a  rubber  stopper  and  the 
tube  is  inverted  several  times ;  on  placing  the  tube  in 
an  upright  position  the  remaining  gas  will  again  collect 
in  the  closed  arm  and  that  absorbed  (CO2)  may  be 
roughly  estimated.  The  gas  still  remaining  will  be 
hydrogen;  if  the  tube  be  inverted  so  that  it  is  forced 
into  the  bulb  the  fact  may  be  ascertained  by  exploding 
it  with  a  lighted  match. 

C.  Animal  Inoculation.  —  Certain  organisms  produce  char- 
acteristic lesions  in  definite  animal  species,  which  are  often  of  great 
value  in  identifying  the  organisms  in  question.  Moreover,  the 
inoculation  of  susceptible  animals  with  contaminated  material 
often  facilitates  the  recovery  of  pathogenic  bacteria  in  pure  cul- 
ture from  the  lesions  produced. 

The  animals  most  frequently  used  for  experimental  purposes 
are  mice,  rats,  guinea  pigs,  and  rabbits,  the  choice  depending 
mainly  on  the  purpose  to  be  served.  Inoculations  are  made  as  a 
rule  by  means  of  a  sterile  hypodermic  syringe  and  needle.  The 
material  used  may  be  a  discharge  such  as  pus,  the  juice  of  organs, 
or  a  culture  of  bacteria ;  if  the  latter  has  been  growing  on  solid 
medium  several  loopfuls  are  emulsified  in  a  small  quantity  of 
sterile  broth  or  physiological  salt  solution. 


62  BACTERIOLOGY  FOR  NURSES 

The  method  of  inoculation  may  be 

(1)  Cutaneous.  —  The  hair  is  removed  by  shaving  from  a  small 

area  of  the  abdomen  or  back  and  the  skin  thoroughly 
cleansed.  A  few  parallel  scratches  are  made  just  deep 
enough  to  draw  blood  and  the  infective  material  rubbed 
in  with  a  sterile  spatula  or  platinum  loop. 

(2)  Intracutaneous.  —  The  hair  is  cut  or  shaved  from  the  part 

to  be  inoculated  and  the  skin  thoroughly  cleansed.  A 
little  of  the  skin  is  pinched  up  between  the  thumb  and 
forefinger  of  the  left  hand,  the  needle  inserted  in  a  slant- 
ing direction,  and  the  inoculation  made.  The  puncture 
may  be  sealed  with  collodion  or  painted  with  iodine. 

(3)  Subcutaneous.  —  The   procedure   is   the  same  as  (2)   save 

that  the  hypodermic  needle  penetrates  beneath  the  skin. 
When  mice  or  rats  are  injected  by  this  method,  the 
inoculation  is  usually  made  near  the  base  of  the  tail. 

(4)  Intraperitoneal.  —  The  hair  is  cut  over  the  lower  part  of 

the  abdomen  and  the  skin  cleansed.  The  entire  thick- 
ness of  the  abdominal  wall  is  pinched  up  between  the 
thumb  and  forefinger  of  the  left  hand,  the  hypodermic 
needle  inserted  and  gently  moved  about  to  be  sure  that 
the  intestine  has  not  been  perforated,  and  the  injection 
made.  On  withdrawing  the  needle  the  puncture  point 
is  painted  with  iodine. 

(5)  Intravenous.  —  The   larger  animals  are  usually  employed 

for  this  method.  If  a  rabbit  is  to  be  inoculated  a  vein 
on  the  outer  margin  of  the  ear  is  chosen,  the  hair  clipped, 
the  skin  cleansed,  and  the  animal  is  held  head  downwards 
for  a  few  seconds  so  that  the  head  may  fill  with  blood 
and  the  vein  become  distended.  The  hypodermic  needle 
is  held  almost  parallel  to  the  ear  and  then  inserted  into  the 
vein.  If  when  the  fluid  is  injected  a  slight  increase  in 
the  lumen  of  the  vein  is  noted  it  will  be  evident  that 
the  injection  has  been  made  into  the  vein ;  if  on  the  other 
hand  a  small  round  swelling  occurs  the  fluid  is  in  the 
surrounding  tissue  and  has  not  entered  the  vein. 


IDENTIFICATION   OF  BACTERIA  63 

(6)  Inoculations  are  occasionally  made  into  the  anterior  cham- 
ber of  the  eye,  the  pleura,  and  the  cranium.  In  certain 
experiments  animals  are  made  to  inhale  dust  or  infected 
spray,  in  others  the  infective  material  may  be  given  in 
food  or  passed  into  the  intestines  by  means  of  a  rubber 
tube. 

Autopsy  on  Dead  Animal.  —  The  autopsy  should  be  made  as 
soon  as  possible  after  death.  If  an  interval  occurs  the  animal  should 
be  kept  at  a  temperature  between  1°  C.  and  4°  C.  For  small 
animals,  the  procedure  is  as  follows :  The  body  is  wiped  with  a  1 
in  20  carbolic  acid  solution  and  stretched  out  back  downwards 
on  a  shallow  metal  trough  having  a  perforation  at  each  end  through 
which  a  tape  or  cord  may  be  passed.  Two  sets  of  sterile  forceps, 
scissors,  and  scalpels  and  several  sterile  Petri  dishes  should  be  ready. 
The  skin  in  the  median  line  of  the  pelvic  region  is  lifted  with  for- 
ceps, and  with  the  scissors  an  incision  is  made  through  the  skin, 
only  upwards  to  the  neck.  From  the  ends  of  this  median  incision 
the  skin  is  cut  outward  toward  each  leg  and  drawn  back  with 
forceps.  If  the  injection  has  been  subcutaneous  the  neighboring 
lymph  nodes  are  examined,  and  if  abnormal  they  are  removed 
with  sterile  scissors  and  forceps  and  placed  in  a  Petri  dish  to  await 
further  examination.  An  incision  is  then  made  through  the  ab- 
dominal wall,  the  diaphragm  is  freed,  and  the  thorax  is  opened  by 
cutting  through  the  ribs  on  both  sides  of  the  sternum.  Films 
and  cultures  are  made  from  any  exudates  and  from  diseased  organs. 
To  obtain  heart  blood  the  tip  of  the  left  ventricle  is  seared  with  a 
red-hot  spatula,  an  incision  is  made  through  the  seared  area,  and 
the  blood  removed  with  a  capillary  pipette  or  a  platinum  loop. 

When  the  examination  is  finished  the  body  should  be  immedi- 
ately burned.  The  instruments  should  be  boiled  in  a  3  per  cent 
sodium  carbonate  solution  for  half  an  hour,  and  the  dissecting 
trough  may  be  allowed  to  stand  twenty-four  hours  in  a  1  in  20 
carbolic  acid  solution. 


CHAPTER  VI 

BACTERIA  IN   NATURAL  PROCESSES   AND 
INDUSTRIES 

WHEN  or  how  life  first  began  on  the  globe  no  one  has  as  yet 
been  able  to  discover.  Nevertheless,  it  is  true  that  it  has  existed 
through  countless  ages  with  no  apparent  decrease  of  vigor !  The 
question  arises  as  to  what  has  become  of  the  waste  products  of 
life  during  these  millions  of  years  and  from  what  limitless  store 
was  the  necessary  food  supply  derived.  The  condition  of  the 
world  is  hardly  conceivable  if  in  the  past  ages  the  dead  bodies  of 
plants  and  animals  had  simply  accumulated  on  the  surface  of  the 
ground.  By  their  very  bulk  they  would  have  so  covered  the  earth 
as  to  afford  no  room  for  the  further  growth  of  plants  or  animals. 
Nor  could  the  soil  furnish  a  food  supply  large  enough  for  the  count- 
less millions  who  have  inhabited  the  earth  and  the  probable  mil- 
lions yet  to  come  without  being  constantly  replenished  from  an 
inexhaustible  store.  The  task  seems  tremendous,  yet  a  large  and 
important  part  of  it  is  performed  by  bacteria. 

First  of  all,  bacteria  act  as  scavengers  in  keeping  the  ground 
in  a  proper  condition  for  the  growth  of  plants  and  animals.  When 
a  tree  dies  and  falls  to  the  ground  an  innumerable  host  of  micro- 
organisms at  once  begin  their  work  of  transformation ;  the  wood 
becomes  softened  and  finally  crumbles  into  a  powdery  mass 
which  sinks  into  the  soil  and  disappears  from  view.  The  body  of 
a  dead  animal  undergoes  a  similar  change ;  the  tissues  decay  rap- 
idly, and  even  the  bones  are  eventually  disintegrated  and  sink  into 
the  soil,  leaving  no  visible  trace.  The  process  of  decomposition  is 
fundamentally  the  same  whether  the  object  be  the  carcass  of  an 
animal,  an  insect,  or  a  tree,  provided  the  necessary  conditions  for 
bacterial  growth  are  present. 

64 


BACTERIA  IN   NATURAL  PROCESSES 


65 


What  has  become  of  what  was  once  an  animal  or  a  tree  ?  The 
answer  is  the  explanation  of  nature's  perpetual  youth:  no  part 
of  that  disintegrating  mass  is  lost ;  all  is  again  utilized  in  one  form 
or  another.  The  greater  part  is  transformed  by  bacteria  into 
substances  that  can  be  used  by  plants  as  food.  The  fact  that 
Nature  works  in  a  cycle,  using  the  same  material  over  and  over 
again,  first  by  the  plant  and  then  by  the  animal  and  then  again 
by  the  plant,  explains  her  seemingly  limitless  supply. 

A  well-known  phase  of  the  interdependence  of  plants  and  ani- 
mals is  the  fact  that  animals  during  respiration  take  in  oxygen 
but  exhale  it  again  in  combination  with  carbon ;  on  the  other  hand, 
plants  draw  into  their  leaves  carbon  dioxide,  retain  the  carbon, 
and  exhale  the  oxygen. 

Nitrogen  Cycle.  —  A  similar  but  more  complex  cycle  occurs  with 
all  the  other  elements  of  plant  and  animal  life.  Substances  are 


.ifion 


Free 
NitroG-er* 


1C 

acterja 


Soil  Nitrates 

FIG.  20. —  Nitrogen  Cycle. 

carried  through  a  series  of  changes  and  in  their  transition  furnish 
the  necessary  energy  for  the  life  work  of  the  individual  whether 
plant  or  animal,  and  finally  they  are  returned  to  approximately 
the  same  form  again,  to  start  once  more  on  their  journey. 

The  food  cycle  is  partially  represented  in  Fig.  20.  The  air 
furnishes  the  plant  with  carbon  dioxide  and  water  and  the  soil  A 
with  the  remaining  ingredients.  Of  the  latter  the  nitrogen  com- 


66  BACTERIOLOGY  FOR  NURSES 

pounds  in  the  form  of  nitrates  (-NOa)  are  the  most  important. 
Potassium,  phosphorus,  and  certain  other  elements  furnish  a  part 
of  the  plant  food,  but  they  are  of  minor  importance  and  for  the 
sake  of  simplicity  may  be  left  out  of  consideration  here.  The 
plant  takes  from  the  soil  and  the  air  the  material  it  requires,  and 
by  means  of  the  energy  furnished  it  by  the  sun's  rays  it  builds 
these  simple  substances  into  more  complex  ones  such  as  sugars, 
starches,  proteins,  and  fats.  This  brings  us  to  step  B.  From 
these  products  of  plant  life  animals  obtain  their  food.  Standing 
at  the  top  of  the  circle  C,  they  are  incapable  of  utilizing  the  simpler 
compounds,  but  are  wholly  dependent  on  the  products  manu- 
factured by  the  plants.  The  complex  food  then  is  eaten  by  ani- 
mals and  becomes  part  of  their  bodies.  As  a  result  of  muscular 
activity  part  of  the  carbon  and  oxygen  is  speedily  converted  into 
carbon  dioxide  and  exhaled  into  the  air.  The  nitrogen  compounds 
are  in  part  reduced  at  once  to  urea  and  excreted  as  such,  while 
most  of  the  remainder  is  retained  to  build  up  body  tissue.  When 
the  body  dies  this  nitrogenous  material  is  far  too  complex  for 
immediate  use  by  the  plants,  and  in  order  to  complete  the  cycle 
it  must  be  reduced  to  simpler  compounds.  It  is  at  this  point  D 
that  bacteria  form  a  link  in  the  chain.  Always  present  in  the 
air,  soil,  and  water,  they  seize  hold  of  any  organic  substance  that 
can  serve  them  as  food  and  break  it  into  simpler  and  simpler  com- 
pounds until  finally  it  is  in  the  right  condition  to  serve  again  as 
plant  food. 

Many  species  take  part  in  this  putrefactive  process.  Bacillus 
proteus,  bacillus  subtilis,  and  the  colon  bacilli  are  amongst  the 
most  important.  Pathogenic  bacteria  are  for  the  most  part  killed 
during  the  early  stages  of  decomposition. 

The  breaking  down  of  the  highly  complex  protein  substances 
into  simpler  and  more  stable  compounds  is  spoken  of  as  minerali- 
zation or  denitrification.  Sometimes  it  happens  that  the  process 
is  carried  so  far  that  the  products  are  too  simple  even  for  plant 
food.  From  decaying  animal  bodies  part  of  the  carbon  is  returned! 
to  the  air  as  carbon  dioxide  and  part  combines  with  the  alkali 
in  the  soil  to  form  carbonates.  The  nitrogen  compounds  that 


BACTERIA  IN  NATURAL  PROCESSES  67 

may  have  been  reduced  to  free  nitrogen  (N),  to  ammonia  (NH3), 
or  to  nitrites  (-NO2),  must  be  built  up  into  nitrates  (-NO3)  before 
the  plants  can  use  them. 

Here  it  is  that  other  species  of  bacteria  E  intervene,  and  this 
time  their  work  is  one  of  construction  rather  than  destruction. 
In  the  soil  everywhere  there  exists  a  class  of  bacteria,  known  as 
nitrifying  bacteria,  which  have  the  power  of  uniting  oxygen  with 
these  simple  compounds.  There  are  apparently  two  distinct 
steps  in  the  process.  In  the  first  stage  ammonia  is  oxidized  to 
nitrous  acid  or  to  nitrites  by  the  nitrosobacteria  or  nitrosomonas. 
These  nitrites  are  somewhat  unstable  and  are  quickly  oxidized 
into  nitrates  by  still  another  species  known  as  nitrobacter.  Whether 
in  the  form  of  nitric  acid  or  nitrates  the  nitrogen  is  now  ready  to 
be  absorbed  by  the  roots  of  the  plant  and  to  start  once  more  on 
its  journey  around  the  food  cycle.  Thus  plants  by  a  constructive 
process  form  the  connecting  link  between  the  soil  and  animal  life, 
and  bacteria,  by  a  reducing  process  sometimes  followed  by  one 
of  synthesis,  complete  the  cycle  of  returning  to  the  soil  again  the 
substances  originally  derived  from  it. 

The  nitrogen  cycle  is  not  quite  complete  at  this  point.  When- 
ever putrefaction  takes  place  some  of  the  nitrogen  escapes  into  the 
air  as  gas  and  is  dissipated.  Apparently  this  portion  has  escaped 
from  the  cycle  since  plants  cannot  extract  free  nitrogen  from  the 
air.  There  are  other  sources  of  apparent  loss  also,  such  as  the 
gradual  draining  of  the  soil  into  streams,  bodies  of  plants  and 
animals  that  fall  into  rivers  and  are  carried  to  sea,  the  sewage 
disposal  of  cities  which  often  discharge  vast  quantities  of  nitrog- 
enous material  into  the  great  lakes  or  the  sea,  the  use  of  nitrog- 
enous compounds  as  explosives.  It  would  seem  in  this  way  that 
large  amounts  of  nitrogen  must  be  irretrievably  lost  from  the 
cycle.  Fortunately,  however,  other  bacteria  are  unceasingly 
at  work  to  redeem  this  supply  which  has  apparently  flown  off  at 
a  tangent.  It  has  been  found  that  soil  entirely  free  from  plants, 
but  containing  certain  species  of  bacteria,  will  slowly  but  surely 
gain  in  the  amount  of  nitrogen  that  it  contains.  That  the  com- 
pounds are  manufactured  by  bacteria  is  certain  because  they  do 


68  BACTERIOLOGY  FOR  NURSES 

not  accumulate  unless  bacteria  are  present.  A  rather  strange 
fact  is  that  this  fixation  of  nitrogen  is  not  accomplished  by  any 
one  species  alone,  but  only  takes  place  when  two.  or  three  are 
acting  together.  The  name  azobacter  is  generally  applied  to  the 
group. 

A  second  method  by  which  bacteria  aid  in  reclaiming  this  dis- 
persed nitrogeri  is  in  combination  with  some  of  the  higher  plants, 
chiefly  beans,  peas,  and  clover.  When  growing  in  soil  that  con- 
tains little  or  no  nitrogen,  these  plants  will,  during  their  growth, 
be  found  to  have  accumulated  a  considerable  store  of  combined 
nitrogen  in  their  tissues.  It  is  evident  that  the  only  possible  source 
of  supply  is  the  nitrogen  of  the  air  that  permeates  the  soil.  When 
a  plant  gains  its  nitrogen  in  this  manner  it  develops  upon  its  roots 
little  protuberances  known  as  root  nodules  or  tubercles,  which  when 
examined  microscopically  are  found  to  be  nests  of  bacteria.  By 
what  process  the  plant  and  the  bacteria  growing  together  succeed 
in  extracting  the  nitrogen  from  the  air  is  not  known.  The  plant 
continues  to  increase  the  store  of  nitrogen  in  its  roots,  stem,  and 
leaves  probably  during  the  whole  of  its  normal  growth.  Finally 
it  dies,  and,  falling  upon  the  ground  becomes  buried.  It  is  imme- 
diately seized  by  the  decomposition  bacteria  and  the  destructive 
changes  already  described  begin.  The  eventual  result  is,  that 
which  seemed  lost  nitrogen  is  once  more  converted  into  nitrates 
and  again  forms  part  of  the  cycle. 

Bacteria,  then,  play  a  threefold  role  in  the  cycle  :  (1)  they  reduce 
complex  nitrogenous  substances  to  simpler  and  more  stable  ones 
such  as  may  be  used  by  plants  for  food ;  (2)  they  build  up  those 
that  are  too  simple  into  suitable  compounds ;  (3)  they  fix  the  nitro- 
gen of  the  air  by  an  unknown  process,  but  by  one  that  makes  it 
again  available  for  the  nourishment  of  plants. 

It  is  important  to  note  that  almost  the  entire  cycle  takes  place 
upon  the  surface  or  in  the  upper  layers  of  the  soil.  A  few  feet 
below  the  surface  there  are  very  few  bacteria ;  consequently  a  car- 
cass buried  deep  or  sewage  placed  too  low  are  not  acted  upon  so 
completely.  At  a  depth  of  six  feet  very  few  organisms  are  found. 

It  is  extremely  difficult  to  determine  the  exact  number  of  bac- 


BACTERIA  IN  NATURAL  PROCESSES  69 

teria  in  any  portion  of  the  soil ;  many  of  them  are  anaerobes  and 
many  require  special  media  for  their  growth.  Of  such  as  can  be 
grown  on  ordinary  media  there  have  been  found  approximately 
100,000  per  gram  in  an  uncultivated  soil,  1,500,000  per  gram  in 
a  garden  soil,  and  115,000,000  per  gram  in  soil  mixed  with  sewage. 
The  actual  numbers,  must  be  infinitely  greater. 

Bacteriological  Examination.  —  For  the  examination  of  sur- 
face soil  a  specimen  may  be  taken  with  a  sterile  spoon  or  tube. 
When  taken  from  a  lower  level  a  special  instrument  is  generally 
used.  The  usual  form  is  that  of  a  drill  with  a  hollow  chamber 
just  above  the  point.  A  sliding  door  to  the  chamber  is  so  arranged 
that  it  can  be  opened  or  closed  by  a  mechanism  controlled  at  the 
handle.  The  chamber  is  first  sterilized  and  then  the  drill  is  forced 
into  the  ground  to  the  desired  depth ;  the  door  of  the  chamber  is 
opened  and  by  a  twisting  movement  the  soil  is  forced  into  the 
chamber ;  the  door  is  then  closed  and  the  drill  removed. 

Before'  removing  the  soil  the  chamber  is  weighed,  then  a  small 
amount  about  the  size  of  a  bean  is  dropped  into  a  flask  containing 
a  liter  of  sterile  water  and  the  chamber  is  again  weighed  to  ascer- 
tain the  quantity  removed.  The  moisture  in  the  flask  is  then 
vigorously  shaken  to  insure  an  even  distribution  of  the  organisms, 
and  the  examination  is  made  in  the  same  manner  as  that  described 
for  water.  Quantities  as  small  as  0.1  c.c.  and  1  c.c.  should  be 
plated  and  cultivated  both  aerobically  and  anaerobically. 

Almost  all  the  bacteria  in  the  soil  are  saprophytes.  The  or- 
ganisms pathogenic  for  man  do  not  find  conditions  favorable 
for  development ;  for  the  most  part  the  temperature  is  too  low,  and, 
further,  they  are  so  crowded  out  by  the  saprophytes  that  they  die 
in  the  struggle  for  existence. 

Pathogenic  Bacteria  Associated  with  the  Soil.  Tetanus  bacilli. 
—  Spores  of  the  tetanus  bacilli  frequently  occur  in  the  soil,  al- 
though it  is  improbable  that  the  organisms  ever  multiply  there. 
Wound  infections  usually  occur  as  a  result  of  contact  with  the  soil 
of  the  object  inflicting  the  wound. 

Anthrax  Bacillus.  —  The  anthrax  bacillus,  like  that  producing 
tetanus,  would  probably  not  continue  to  exist  in  the  soil  were  it 


70  BACTERIOLOGY  FOR  NURSES 

not  for  its  resistant  spores.  Its  presence  there  is  a  greater  menace 
to  animals  than  to  man. 

Malignant  Edema.  —  Wound  infections  with  the  bacillus  of 
malignant  edema  occur  which  give  rise  to  extensive  hemorrhagic 
edema.  The  bacillus  is  an  anaerobe  and  occurs  in  the  upper 
layer  of  the  soil. 

Welch's  Gas  Bacillus  or  B.  aerogenes  capsulatus.  —  This  or- 
ganism occurs  in  the  intestines  of  man  and  animals  and  in  the  soil. 
When  introduced  into  wounds  it  may  produce  suppuration  and  a 
great  amount  of  gas.  In  the  majority  of  cases  it  leads  to  no  harm, 
yet  in  a  small  percentage  it  causes  one  of  the  most  rapidly  fatal 
infections  known. 

Typhoid  Bacillus. — Typhoid  bacilli  may  find  their  way  into  the 
soil  with  human  excreta.  Multiplication,  however,  rarely  takes 
place  there.  As  a  rule  they  do  not  live  more  than  a  month  unless 
the  ground  is  frozen,  in  which  case  their  life  may  be  prolonged 
to  several  months.  The  chief  danger  so  far  as  typhoid  bacilli 
are  concerned  is  the  washing  by  heavy  rains  of  human  excreta 
that  has  been  deposited  on  the  ground  into  a  stream  used  for 
drinking  purposes,  or  drainage  through  the  soil  into  a  near-by 
well. 

Cholera  Spirillum.  —  Cholera  spirilla  too  may  be  deposited 
upon  the  ground  in  human  feces;  they  live  only  a  very  short 
time  there  and  are  not  likely  to  regain  entrance  into  the  human 
body  except  by  means  of  drinking  water. 

BACTERIA  IN   THE   INDUSTRIES 

The  ability  of  bacteria  to  produce  decomposition  is  the  basis 
of  several  industries.  Certain  of  these  depend  upon  the  result 
of  bacterial  fermentation,  others  exist  to  prevent  it.  Many  of 
them  were  in  operation  long  before  their  intimate  relation  to  bac- 
terial activity  was  known.  In  some  cases  the  original  methods 
are  still  employed,  though  modified  somewhat  by  the  use  of  pure 
cultures  or  an  increased  knowledge  of  antiseptics  and  germicides. 

Preservation  of  Food.  —  The  ceaseless  energy  of  bacteria  and 


BACTERIA   IN   INDUSTRIES  71 

their  presence  everywhere  makes  it  impossible  to  preserve  meat 
and  fruit  for  more  than  a  few  days  without  applying  special 
methods.  This  fact,  coupled  with  the  necessity  for  conserving 
a  supply  for  the  months  when  such  foods  are  scarce,  has  given 
rise  to  one  of  the  most  important  industries.  Canning  of  meats 
and  fruit  is  simply  preserving  them  from  the  attack  of  micro- 
organisms ;  heating  kills  all  the  bacteria  present,  and  hermetically 
sealing  prevents  others  from  gaining  access. 

The  process  of  canning  was  practised  as  early  as  1804.  Monsieur 
Appert  of  Paris  found  that  food  in  sealed  vessels  would  keep  in- 
definitely if,  after  being  sealed,  the  containers  were  kept  for  one 
hour  in  boiling  water.  His  method  is  still  employed  except  that 
after  an  interval  of  a  day  a  second  heating  is  now  given  to  destroy 
forms  that  might  have  been  in  a  spore  stage  on  the  first  day. 
Canning  is  really  a  practical  application  of  fractional  sterilization. 

Drying  is  one  of  the  oldest  and  simplest  methods  of  preserving 
food  from  bacterial  attack.  Exposure  to  sun  and  air  deprives  the 
substances  of  their  moisture  and  consequently  renders  them 
unsuitable  for  bacterial  growth.  Smoke-dried  meats  in  addition 
to  losing  their  moisture  are  impregnated  with  antiseptic  substances 
such  as  creosote,  which  are  present  in  varying  amounts  in  wood 
smoke.  It  has  been  found,  however,  that  smoking  cannot  be 
depended  upon  to  destroy  disease-producing  organisms  in  con- 
taminated meats. 

The  addition  of  chemical  substances  that  prevent  the  growth 
of  bacteria  has  long  been  practiced ;  vinegar  is  a  familiar  example. 
Meat  placed  in  brine  and  fruit  in  a  thick  sugar  sirup  are  preserved, 
because  the  density  of  the  solution  being  greater  than  that  of  the 
bacterial  cell,  water  is  drawn  from  the  microorganism  rather  than 
supplied  to  it.  Strongly  bactericidal  substances  are  sometimes 
used,  such  as  borax,  salicylic  acid,  and  formaldehyde.  They  may 
accomplish  the  purpose  for  which  they  were  added,  but  except  in 
very  small  amounts  they  are  injurious  to  the  consumer. 

The  cold-storage  method  of  food  preservation  has  as  its  prin- 
ciple the  inhibition  of  bacterial  growth  by  a  low  temperature. 
Bacterial  multiplication  ceases  at  a  few  degrees  above  freezing 


72  BACTERIOLOGY  FOR  NURSES 

point ;  hence  refrigeration  is  an  excellent  preservative.  Bacterial 
activity  is  thus  a  reason  for  the  existence  of  the  ice  industry  and 
the  manufacture  of  refrigerators. 

Occasionally  bacteria  are  intentionally  allowed  to  decompose 
food  up  to  a  certain  limit.  The  so-called  gamy  flavor  of  meat 
is  due  to  the  first  stage  of  decomposition.  Sauerkraut  is  another 
example  of  food  expressly  allowed  to  ferment.  The  special  flavors 
produced  by  bacteria  in  the  preparation  of  butter  and  cheese  are 
other  instances. 

Vinegar  Making.  —  The  first  step  in  the  process  of  vinegar  mak- 
ing is  brought  about  by  yeast  cells.  By  their  ability  to  ferment 
grape  sugar  they  produce  alcohol.  The  next  stage  is  accomplished 
by  bacteria  which  cause  the  alcohol  to  unite  with  oxygen,  thus 
producing  acetic  acid  or  vinegar.  Oxidation  of  alcohol  into  vine- 
gar can  be  brought  about  by  a  chemical  process,  but  it  is  impracti- 
cable on  a  large  scale. 

One  of  the  usual  methods  employed  is  to  add  to  a  weak  solution 
of  cider  a  small  quantity  of  vinegar.  After  a  short  time  a  thick, 
felted  scum  forms  on  the  surface  of  the  alcohol.  The  scum  is  a 
mass  of  bacteria  spoken  of  as  the  "mother  of  vinegar,"  which  in 
some  way  causes  the  oxygen  of  the  air  to  unite  with  the  alcohol. 
After  the  amount  of  acetic  acid  reaches  a  certain  percentage 
bacterial  action  stops  and  no  more  acid  is  produced,  even  though 
there  be  alcohol  remaining.  It  was  at  first  thought  that  only 
one  species  of  bacteria  was  able  to  produce  this  fermentation. 
Later  study  has  shown  that  several  different  kinds  have  the  power ; 
most  of  them  have  a  common  characteristic  in  that  they  grow  in 
long  filaments  without  any  trace  of  division. 

A  rapid  method  of  vinegar  manufacture  is  carried  on  by  filling 
high  cylinders  about  three  fourths  full  with  wood  shavings  which 
have  been  soaked  in  warm  vinegar.  Weak  alcohol  is  then  poured 
in,  and  as  it  slowly  passes  over  the  shavings  it  is  oxidized  into 
acetic  acid. 

Occasionally  the  fermentation  does  not  proceed  in  a  satisfactory 
manner ;  other  species  find  their  way  into  the  fermenting  liquid 
and  produce  undesirable  substances  which  give  it  a  totally  dif- 


73 

ferent  flavor.  By  degrees  a  more  scientific  method  is  being 
adopted.  It  is  gradually  becoming  the  custom  to  heat  the  alcohol 
and  then  add  the  desired  bacteria  in  pure  culture. 

Maceration  Industries.  —  The  separation  of  linen  from  the  flax 
stem  is  a  process  usually  brought  about  by  bacterial  activity. 
The  valuable  linen  fibers  and  the  coarser  wood  are  so  bound  to- 
gether by  a  cementing  substance  that  it  is  seldom  possible  to 
separate  them  by  mechanical  means.  In  order  to  decompose 
this  binding  material  several  methods  may  be  employed,  the  prin- 
ciple of  each  being  to  subject  the  stems  to  suitable  heat  and  mois- 
ture to  encourage  bacterial  growth.  A  fermentation  is  thus  started 
which  softens  the  gummy  substance  holding  the  fibers  together 
and  permits  their  separation.  This  "  water-retting  "  process  is 
supposedly  brought  about  by  anaerobic  bacteria. 

The  same  principle  is  applied  in  the  manufacture  of  jute  and 
hemp  and  in  the  preparation  of  cocoanut  fiber.  In  the  tanning 
of  leather,  the  preparation  of  sponges,  and  the  curing  of  tobacco 
bacteria  also  play  a  large  part. 


CHAPTER  VII 

BACTERIOLOGICAL  EXAMINATION  OF  WATER  AND 

SEWAGE 

NATURAL  waters  are  usually  considered  in  three  classes  accord- 
ing to  their  location ;  namely,  rain  water,  ground  water,  and  surface 
water. 

Practically  all,  from  whatever  source,  contain  bacteria,  the  num- 
ber and  kind  varying  under  different  conditions.  Rain  water 
contains  comparatively  few  excepting  the  first  shower,  which 
washes  the  air  and  brings  down  most  of  the  floating  dust  particles 
and  bacteria  in  its  fall. 

Ground  waters,  which  include  springs,  shallow  wells,  and  deep 
artesian  wells,  rank  next  from  the  standpoint  of  bacteriological 
purity.  The  water  as  it  percolates  through  the  soil  gradually 
leaves  behind  its  bacterial  content  in  the  upper  layers  of  the  ground 
and  finally  emerges  in  a  spring  or  well  almost  germ  free.  This  is 
especially  true  of  artesian  wells  and  springs.  Shallow  wells  are 
more  liable  to  variation.  Unclean  surroundings,  privy  vaults, 
or  barns  placed  in  such  a  position  that  drainage  may  take  place 
in  the  direction  of  the  well  may  lead  to  contamination  of  the  water 
and  a  consequent  increase  in  the  number  of  bacteria. 

Surface  waters  include  streams,  rivers,  ponds,  and  lakes,  and 
these  of  all  natural  supplies  contain  the  most  bacteria  on  account 
of  the  exposure  to  contamination  to  which  they  are  subjected. 
During  heavy  rains  soil  washed  down  from  the  banks  of  rivers  or 
streams  supply  an  additional  number  of  bacteria ;  wind  currents 
and  waves  stirring  up  the  bottom  mud  may  bring  up  bacteria 
that  have  been  sedimented ;  sewage  and  trade  wastes  from  near-by 
towns  may  add  enormously  to  the  bacterial  content. 

74 


EXAMINATION  OF  WATER  AND  SEWAGE        75 

Relative  Purity.  —  An  arbitrary  standard  of  relative  purity  is 
almost  impossible  to  fix.  Several  hundred  bacteria  per  cubic  centi- 
meter might  be  normal  in  a  river  water,  whereas  the  same  number 
found  in  well  water  would  immediately  arouse  suspicion.  Accord- 
ing to  certain  authorities  water  containing  less  than  100  bacteria 
per  c.c.  is  presumably  uncontaminated  by  surface  drainage,  one 
with  500  bacteria  per  c.c.  is  open  to  suspicion,  one  with  1000 
per  c.c.  is  presumably  contaminated  by  sewage  or  surface  drainage. 
A  practical  classification  from  a  sanitary  point  of  view  is  as  follows : 
(1)  good,  as  determined  by  bacteriological  and  chemical  analyses, 
physical  inspection,  and  a  sanitary  survey  of  the  watershed ;  (2) 
contaminated,  if  organic  waste  of  either  animal  or  vegetable  origin 
be  present  (a  contaminated  water  is  suspicious  but  not  necessarily 
dangerous) ;  (3)  infected,  if  the  water  contains  specific  organisms 
causing  disease. 

Significance  of  the  Presence  of  Colon  Bacilli.  —  Enumeration 
of  bacteria  gives  an  approximate  idea  of  the  degree  of  pollution 
with  organic  material,  but  it  gives  no  idea  of  the  kind  of  bacteria 
present.  Unfortunately  there  is  no  reliable  method  whereby 
pathogenic  organisms  such  as  the  typhoid  and  dysentery  bacillus 
can  be  isolated  from  water  with  any  degree  of  certainty,  even 
though  it  is  actually  known  that  the  organisms  are  or  have  been 
present  because  of  cases  of  disease  which  have  developed  from 
drinking  it.  The  bacteria  may  be  present  in  such  small  numbers 
that  though  an  ordinary  tumbler  of  water  might  contain  sufficient 
to  cause  infection  it  would  be  a  rare  chance  if  one  or  two  of  them 
should  be  in  the  small  quantity  taken  for  examination.  Another 
likely  reason  for  failure  is  the  fact  that  they  do  not  live  many 
days  in  water.  It  is  known,  however,  that  certain  organisms, 
such  as  the  colon  bacilli,  which  are  normally  present  in  the  intes- 
tines, have  a  longer  life  in  water  than  those  which  are  present  only 
in  diseased  conditions.  Moreover,  it  is  practically  sure  that  all 
the  pathogenic  organisms  which  give  rise  to  water-borne  infections 
find  their  way  into  the  water  supply  by  means,  of  intestinal  dis- 
charges from  human  beings.  Under  these  conditions  it  is  practi- 
cally safe  to  assume  that  the  absence  of  the  colon  bacilli  in  water 


76  BACTERIOLOGY  FOR  NURSES 

means  the  absence  of  pathogenic  bacteria  except  in  such  extremely 
rare  cases  as  contamination  by  urine.  The  presence  of  the  colon 
bacillus  does  not  necessarily  signify  danger,  but  it  does  mean  pollu- 
tion with  fecal  discharges.  Deep  well  water  should  be  condemned 
if  any  colon  bacilli  are  found  in  it.  On  the  other  hand,  surface 
water  may  contain  one  colon  bacillus  per  c.c.  without  the  presence 
of  pathogenic  organisms  being  suspected,  particularly  if  it  is  known 
to  drain  an  inhabited  area.  The  fact  that  the  colon  bacillus  is 
found  in  the  feces  of  animals  makes  it  difficult  to  determine  whether 
pollution  is  of  animal  or  human  origin.  A  fresh  hillside  stream 
may  contain  colon  bacilli  brought  to  it  by  rain  washings  from 
manured  fields  through  which  it  passes  or  by  a  stray  horse  or  cow. 
Any  water,  however,  containing  ten  colon  bacilli  per  c.c.  is  usually 
considered  as  decidedly  polluted  and  unsafe  for  human  consump- 
tion. 

Generally  speaking,  no  single  test  should  be  relied  on  alone,  al- 
though the  colon  test  surpasses  all  others  in  delicacy  in  determin- 
ing pollution.  An  inspection  of  the  surroundings,  a  chemical  and 
bacteriological  examination,  are  all  necessary  for  complete  infor- 
mation. 

Bacteriological  Analysis.  —  In  the  bacteriological  examination 
of  water  two  lines  of  inquiry  are  generally  followed.  First,  the 
approximate  number  of  bacteria  per  c.c.  is  estimated,  and  second, 
the  presence  or  absence  of  the  colon  bacillus  is  determined,  and 
if  present  in  what  number  per  c.c.  it  occurs. 

As  in  the  case  of  soil,  so  also  in  a  given  sample  of  water,  it  is 
impossible  to  determine  the  exact  number  of  living  organisms  pres- 
ent. This  fact,  however,  does  not  prevent  a  numerical  estimation 
from  being  of  value.  An  approximate  idea  of  the  number  present 
can  be  obtained,  and  also  certain  inferences  of  importance  can 
be  drawn  by  comparing  the  results  obtained  by  different  methods. 
For  example,  if  a  moderate  number  of  bacteria  develop  at  20°  C. 
and  very  few  at  37°  C.  the  water  may,  from  a  sanitary  standpoint, 
be  comparatively  pure.  If,  however,  the  condition  is  reversed  and 
many  colonies  appear  upon  agar  plates  incubated  at  37°  C.  and 
few  at  20°  C.,  then  the  majority  of  the  organisms  present  may  be 


EXAMINATION  OF  WATER  AND  SEWAGE        77 

regarded  as  accustomed  to  the  same  temperature  found  within 
the  animal  body  and  the  water  consequently  as  suspicious. 

Collecting  Samples.  —  Care  must  be  taken  that  a  sample  is 
representative.  If  taken  from  a  tap  or  pump  the  water  in  the  pipe 
must  first  be  run  off  to  obviate  any  effect  the  metal  may  have  had ; 
if  from  a  lake  or  pond  the  surface  scum  or  bottom  mud  should  be 
avoided  or  both  may  be  examined  separately ;  if  from  a  brook  the 
specimen  should  be  taken  some  distance  from  the  bank. 

The  container  must  of  course  be  sterile  and  the  test  made  as 
soon  as  possible,  for  an  increase  or  a  decrease  in  the  number  of 
bacteria  begins  immediately.  If  a  short  delay  is  unavoidable 
the  sample  should  be  kept  on  ice  at  a  temperature  of  5°  C. 

Technique  for  Quantitative  Analysis.  —  The  sample  is  vigorously 
shaken  about  twenty-five  times  and  then  by  means  of  a  sterile 
graduated  pipette  1  c.c.,  0.1  c.c.,  and  0.01  c.c.  are  placed  respec- 
tively in  three  sterile  Petri  dishes  previously  marked  with  the 
amount  to  be  received.  Immediately  a  tube  of  liquid  agar  cooled 
to  40°  C.  is  poured  into  each  dish  and  the  water  and  agar  are  thor- 
oughly mixed  by  a  rotary  movement  of  the  dish  before  the  agar 
solidifies.  The  test  should  be  made  in  duplicate,  one  set  of  plates 
being  placed  in  the  incubator  at  37°  C.  for  twenty-four  hours  and 
the  other  kept  in  the  dark  at  room  temperature  for  forty-eight 
hours.  Usually  the  number  of  colonies  developing  at  room  tem- 
perature is  far  greater  than  that  developing  at  37°  C.  The  colonies 
are  counted  in  the  manner  already  described. 

Presumptive  Test  for  B.  Coli.  —  To  determine  the  presence  of 
the  colon  bacillus  a  medium  such  as  the  Conradi-Drigalsky  is 
employed,  and  the  plating  procedure  already  indicated  is  done 
in  triplicate,  to  the  third  series  of  plates  being  added  the  colored 
medium  instead  of  the  plain  agar.  After  twenty-four  hours' 
incubation  if  colon  bacilli  are  present  in  the  amount  of  water  tested 
red  characteristic  colonies  will  appear  which  stand  out  well  against 
the  blue  background. 

A  second  test  is  made  by  inoculating  fermentation  tubes  con- 
taining colored  lactose  peptone  water  with  varying  amounts  of 
water :  10  c.c.,  1  c.c.,  and  0.1  c.c.  After  incubation  at  37°  C.  for 


78  BACTERIOLOGY  FOR  NURSES 

forty-eight  hours  those  tubes  showing  a  color  change  and  the  pro- 
duction of  gas  are  presumed  to  contain  the  colon  bacillus.  If, 
for  example,  gas  appears  in  the  tubes  containing  10  c.c.  and  1  c.c. 
of  water  and  not  in  the  tube  containing  0.1  c.c.  it  is  assumed  that 
the  water  contained  at  least  one  colon  bacillus  per  c.c.  The  in- 
terpretation of  the  results  by  the  plate  method  is  evident.  The 
number  of  red  colonies  developing  on  the  plate  containing  1  c.c. 
of  water  would  give  the  number  of  B.  coli  present  per  c.c. ;  if 
these  should  be  too  numerous  to  count  or  if  the  color  of  the  entire 
medium  be  changed,  then  the  colonies  on  the  plate  containing  0.1 
c.c.  of  water  should  be  counted  and  the  number  multiplied  by  ten 
to  give  the  total  number  per  c.c. 

It  should  be  remembered  that  the  above  are  only  presumptive 
or  partial  tests  for  the  colon  bacillus,  and  although  fairly  reliable 
and  of  value  for  routine  examination  other  tests  are  necessary 
before  an  organism  can  with  certainty  be  said  to  be  the  colon 
bacillus. 

Determination  Test.  —  In  order  to  determine  beyond  doubt 
that  B.  coli  has  caused  the  fermentation  of  lactose  in  the  tubes 
a  loopful  of  the  culture  is  streaked  on  plates  containing  solidified 
Conradi  medium,  and  after  twenty-four  hours'  incubation  a  typical 
red  colony  is  fished  and  incubated  in  a  tube  of  broth.  At  the 
end  of  from  twelve  to  twenty-four  hours'  incubation  about  0.1  c.c. 
of  the  broth  culture  is  pipetted  into  the  following  media,  each 
of  which  gives  a  characteristic  reaction  when  used  for  the  culti- 
vation of  the  colon  bacillus:  gelatin,  absence  of  liquefaction; 
neutral  red  lactose  peptone  water,  production  of  acid  and  gas ;  milk, 
production  of  acid  and  coagulation  of  the  protein ;  peptone  solu- 
tion, production  of  indol. 

Sewage  Streptococci.  —  In  addition  to  the  many  different 
bacilli  normally  present  in  the  intestines  there  are  also  certain 
cocci  often  spoken  of  as  sewage  streptococci.  They,  too,  produce 
pink  colonies  on  Conradi  medium.  They  are  much  smaller,  how- 
ever, than  those  of  the  colon  bacillus  and  can  easily  be  differenti- 
ated. They  are  not  hardy  and  quickly  die  in  water ;  thus  their 
presence  represents  recent  pollution. 


EXAMINATION  OF  WATER  AND  SEWAGE        79 

Isolation  of  the  Typhoid  Bacillus.  —  So  many  difficulties  attend 
the  search  for  the  typhoid  bacillus  in  water  that  it  is  rarely  at- 
tempted except  in  experimental  research.  Under  ordinary  cir- 
cumstances the  organisms  do  not  multiply  in  water ;  they  rarely 
live  longer  than  seven  days  in  cold  water  and  even  a  shorter  period 
when  it  is  warm. 

Several  methods  have  been  devised  for  the  isolation  of  the 
typhoid  bacillus,  one  of  which  is  the  addition  of  large  quantities 
of  water  to  the  same  volume  of  double-strength  broth  containing 
a  substance  known  to  inhibit  the  growth  of  saprophytic  organisms 
without  having  an  injurious  effect  on  the  typhoid  bacillus.  After 
twenty-four  hours  pour  plates  are  made,  employing  one  of  the 
special  media,  such  as  the  Conradi-Drigalsky.  It  is  exceedingly 
rare,  however,  that  the  organism  is  isolated.  Water  is  more  often 
condemned  on  circumstantial  evidence  than  on  the  actual  finding 
of  the  typhoid  bacillus. 

Cholera  Spirillum.  —  The  isolation  of  the  cholera  spirillum  from 
water  is  somewhat  less  difficult.  It  occurs  in  much  greater  num- 
bers in  the  excreta  of  cholera  patients  than  does  the  typhoid 
bacillus  in  the  feces  of  those  suffering  from  typhoid  fever.  Koch, 
the  discoverer  of  the  cholera  spirillum,  suggested  a  practical  method 
which  has  proved  of  value.  The  water  to  be  examined  is  itself 
converted  into  medium  by  dissolving  in  each  liter  ten  grams  of 
peptone  and  sufficient  sodium  carbonate  to  make  it  slightly  alka- 
line. The  mixture  is  incubated  at  37°  C.  from  sixteen  to  twenty 
hours,  after  which  gelatin  or  agar  plates  are  streaked  with  the 
surface  growth.  If  cholera  spirilla  are  present  characteristic 
colonies  with  irregular  margins  will  develop  which  will  agglutinate 
with  specific  cholera  serum. 

Purification  of  Water.  —  Nature  has  various  methods  of  her  own 
for  the  purification  of  water.  Enormous  quantities  of  sea  water 
and  marsh  water  are  being  constantly  evaporated  and  then  returned 
in  the  form  of  rain  in  a  practically  pure  state.  Streams  tend  to 
become  purer  in  their  flow ;  organic  matter  is  gradually  oxidized, 
thus  diminishing  the  bacterial  food  supply.  Microscopic  animals 
such  as  protozoa  feed  upon  bacteria,  and  they  in  turn  serve  as 


80  BACTERIOLOGY  FOR  NURSES 

food  for  rotifers  and  Crustacea.  Dilution  plays  an  important 
role  in  that  a  small  amount  of  infection  in  a  lake  or  river  is  soon 
so  diluted  as  to  practically  become  lost.  A  slow-moving  river 
is  purified  much  in  the  same  way  that  snow  clears  the  air:  the 
particles  of  mud  which  are  constantly  settling  enmesh  the  bac- 
teria in  their  fall  and  carry  them  down  to  the  bottom,  where  they 
soon  die. 

Boiled  Water.  —  So  far  as  water-borne  infections  are  concerned 
boiling  renders  water  safe.  Typhoid  and  dysentery  bacilli  and 
cholera  spirilla  are  killed  even  at  a  lower  temperature.  Holding 
for  twenty  minutes  at  60°  C.  or  a  few  minutes  at  70°  C.  is  sufficient 
to  destroy  them. 

The  principal  methods  employed  for  the  purification  of  water  on 
a  large  scale  are  (1)  storage,  (2)  filtration,  (3)  addition  of  a  chemi- 
cal. In  some  cities  two  or  even  all  the  methods  are  combined. 

Storage.  —  Several  of  Nature's  methods  are  applied  in  this  form 
of  purification;  namely,  time,  oxidation,  dilution,  sedimentation, 
etc.  The  growth  of  algae  and  decomposition  of  organic  matter 
sometimes  gives  to  water  stored  in  an  open  reservoir  a  disagree- 
able taste  and  odor.  That  may  be  obviated,  however,  by  the  use 
of  a  closed  reservoir. 

Filtration.  —  Two  forms  of  filters  are  in  general  use  for  public 
water  supplies  :  slow  sand  filters  and  mechanical  filters. 

A  slow  sand  filter  consists  of  a  large  shallow  reservoir  with 
underdrain  pipes  and  containing  five  or  six  feet  of  filtering  material 
of  graded  size,  beginning  at  the  bottom  with  broken  stone  or  gravel 
and  finishing  with  an  upper  layer  of  fine  sand.  The  water  passes 
through  the  filter  very  slowly  from  above  downwards,  and  in  its 
passage  almost  all  the  bacteria  and  fine  particles  are  strained  out. 
The  process  is  not  merely  a  simple  straining ;  its  efficiency  is  due 
rather  to  bacterial  activity.  The  spaces  between  the  finest  sand 
are  enormous  compared  to  the  size  of  bacteria,  and  yet  99  per  cent 
of  bacteria  do  not  pass  beyond  the  upper  layer.  What  really 
happens  is  that  the  microorganisms  resting  upon  the  surface  grow 
and  form  gelatinous  masses  which  adhere  to  the  particles  of  sand 
and  gradually  close  up  the  interstices.  This  continuous  carpet- 


EXAMINATION  OF  WATER  AND  SEWAGE        81 

like  mass  effectively  holds  back  the  bacteria.     Thus  their  removal 
is  largely  a  biological  process  due  to  the  bacteria  themselves. 

The  action  of  a  mechanical  filter  is  strictly  a  straining.  A  chemical 
coagulant  is  added,  generally  sulphate  of  aluminium,  and  the  water 
is  passed  rapidly  through  a  layer  of  sand.  The  coagulant  clears 
the  water  much  as  the  white  of  an  egg  clears  coffee.  Bacteria 
are  enmeshed  and  deposited  on  the  surface  of  the  sand,  forming 
thus  an  artificial  inorganic  carpet  in  place  of  the  natural  organic 
one  of  the  slow  sand  filter  bed.  Mechanical  filtration  is  a  compar- 
atively quick  process  and  especially  suitable  for  turbid  waters 
containing  much  clay ;  its  action  is  somewhat  less  uniform  than 
slow  sand  filtration.  It  removes  from  95  per  cent  to  99  per  cent  of 
bacteria. 

Household  filters  of  the  ordinary  type  cannot  be  relied  upon  to 
make  infected  water  safe.  They  are  serviceable  in  rendering  a 
turbid  water  clear,  but  they  should  not  be  depended  upon  for  more 
than  that. 

Addition  of  Chemicals.  —  Ozone  is  an  effective  purifier  of  water, 
but  its  use  is  limited  in  that  it  does  not  clarify,  and  the  expense 
of  producing  it  is  comparatively  large. 

Chlorinated  Lime,  Chloride  of  Lime  or  Bleaching  Powder.  — 
The  germicidal  action  is  due  to  liberated  chlorine  'which  acts  on 
the  water,  setting  free  nascent  oxygen.  So  effective  is  chlorinated 
lime  that  one  part  per  million  parts  of  water  will  destroy  99  per 
cent  of  the  bacteria  in  water  containing  little  organic  material. 
It  does  not  clarify  water  nor  remove  discoloration,  but  it  is  a  cheap, 
efficient,  and  harmless  method  and  is  widely  used. 

Copper  Sulphate.  —  It  was  first  claimed  that  the  addition  of 
copper  sulphate  in  small  amounts  to  water  would  destroy  both 
the  algae  which  produce  objectionable  tastes  and  odors  and  the 
pathogenic  microbes.  Later  it  was  found  that  while  even  in  great 
dilution  it  destroys  algse  and  many  microorganisms  it  has  little 
effect  upon  typhoid  and  dysentery  bacilli.  It  is  generally  used 
in  the  proportion  of  one  tenth  to  one  quarter  part  per  million  parts 
of  water.  The  copper  combines  with  the  bodies  of  the  organisms 
and  both  settle  to  the  bottom  as  a  sediment. 


82  BACTERIOLOGY  FOR  NURSES 

Ultraviolet  Rays.  —  Exposure  of  a  clear  water  to  ultraviolet 
rays  has  a  speedy  effect  on  bacteria.  Most  of  the  vegetative  forms 
are  killed  in  from  ten  to  twenty  seconds ;  a  longer  time,  however, 
is  necessary  if  the  water  is  turbid.  After  preliminary  rough  fil- 
tration to  remove  coarse  particles  the  water  supply  of  Marseilles 
passes  a  quartz  tube  mercury  arc  lamp  three  times.  It  is  claimed 
that  between  98  and  99  per  cent  of  the  bacteria  present  are  de- 
stroyed during  the  process. 

Sewage  Purification.  —  The  role  which  bacteria  play  in  the 
disintegration  and  consequent  purification  of  sewage  is  an  impor- 
tant one,  even  though  the  details  of  the  process  are  somewhat 
obscure. 

The  treatment  of  sewage  may  consist  of  one,  two,  or  all  of  the 
following  processes:  (1)  screening  or  removing  the  larger  sub- 
stances that  might  injure  filters,  etc.  Screens  may  vary  all  the 
way  from  gratings  of  iron  bars  to  fine  ones  of  wire  cloth.  The 
material  screened  is  pressed  and  burned  under  a  boiler  or  buried 
in  land  and  the  effluent  passed  into  (2)  a  sedimentation  tank. 
Several  different  types  of  tanks  have  been  devised  for  the  purpose, 
in  all  of  which  the  underlying  principle  is  the  disintegration  by 
bacteria  of  the  organic  material  present.  From  the  sedimentation 
tank  the  sewage  is  carried  to  (3)  a  filter  bed,  usually  composed  of 
some  form  of  porous  material  such  as  coke  or  brick,  through  which 
it  percolates  to  underdrains  below.  The  final  process  (4)  is  the 
removal  or  destruction  of  bacteria  in  the  effluent.  Chlorinated 
lime  is  one  of  the  best  substances  for  the  purpose;  about  fifty 
pounds  per  million  gallons  for  good  effluents  will  destroy  from 
95  to  99  per  cent  of  the  bacteria. 

Methods  of  bacteriological  examination  to  determine  the  effi- 
ciency of  a  purification  plant  are  the  same  as  those  described  for 
the  examination  of  water  except  that  smaller  quantities  are  used. 
In  a  sewage  effluent  as  in  water  the  absence  of  the  colon  bacillus 
is  regarded  as  an  indication  of  its  harmlessness. 


CHAPTER  VIII 
MILK 

BECAUSE  milk  is  one  of  the  most  valuable  articles  of  diet,  and 
above  all  because  it  is  an  indispensable  food  for  infants  and  young 
children,  its  purity  from  a  bacteriological  standpoint  is  of  the  great- 
est importance.  As  a  rule,  however,  it  contains  more  bacteria 
than  any  other  article  of  food ;  frequently  many  more  than  are 
found  in  sewage.  For  the  most  part  the  bacteria  which  find  their 
way  into  milk  are  saprophytes,  but  even  so,  their  presence  in  large 
numbers  is  by  no  means  desirable  in  a  food  for  infants. 

When  first  secreted  in  the  udder  of  a  normal  cow  milk  is  prac- 
tically germ  free.  It  is  impossible,  however,  by  ordinary  dairying 
methods  to  obtain  it  in  such  a  pure  condition.  Bacteria  from  the 
air  and  surroundings  find  their  way  into  the  milk  ducts,  and  as  a 
little  milk  always  remains  there  from  the  previous  milking  they 
find  exactly  the  conditions  they  require :  food,  moisture,  and  a 
suitable  temperature,  and  they  begin  to  multiply  rapidly.  By  the 
next  milking  they  are  abundant,  and  the  first  milk  drawn  washes 
them  into  the  milk  pail,  where  they  continue  to  grow.  The  milk 
receives  an  additional  supply  from  all  the  objects  with  which  it 
comes  in  contact.  The  hands  of  the  milker,  the  air  through  which 
it  passes,  and  the  pail  into  which  it  falls  add  their  quota.  The 
hairs  of  the  cow  and  particles  of  manure  which  may  drop  into  the 
pail  furnish  more.  Generally  the  farmer  makes  an  attempt  to  re- 
move the  coarser  dirt  by  straining  the  milk  through  a  cloth.  That 
process,  however,  does  not  affect  the  bacteria  present. 

Even  a  moderate  degree  of  cleanliness  has  an  appreciable  effect 
upon  the  bacterial  content.  A  clean  barn  in  which  to  milk,  clean 
pails  with  small  openings,  clean  hands,  and  a  clean  condition  of  the 

83 


84 


BACTERIOLOGY  FOR  NURSES 


cow's  udder  and  flanks  will  mean  a  considerably  lower  bacterial 
count.  Milk  obtained  by  the  cleanest  methods  may  contain 
only  a  few  hundred  bacteria  per  c.c.  when  drawn ;  collected  with 
less  care  it  may  contain  several  thousand,  and,  unless  promptly 
cooled,  the  number  soon  mounts  to  millions.  An  excessive  number 
of  bacteria,  therefore,  is  an  indication  that  milk  is  dirty  or  old  or 
that  care  has  not  been  taken  to  keep  it  cool.  The  following  table 
illustrates  these  points  well. 


TABLE  I1 

Milk  collected  under  the  best  conditions  possible.      Bacterial 
content  at  commencement  of  test  3000  per  c.c. 


KEPT  AT 

24  HRS. 

48  HRS. 

96  HRS. 

168  HRS. 

o°c. 

2,400 

2,100 

1,850 

1,400 

4°C. 

2,500 

3,600 

218,000 

4,209,000 

10°  C. 

11,500 

540,000 

300,000,000 

1,000,000,000 

20°  C. 

450,000 

500,000,000 

TABLE  II 

Milk  collected  under  ordinary  conditions.     Bacterial  content 
at  commencement  of  test  30,000  per  c.c. 


KEPT  AT 

24  HRS. 

48  HRS. 

96  HRS. 

168  HHS. 

o°c. 

30,000 

27,000 

24,000 

19,000 

4°C. 

38,000 

56,000 

4,300,000 

38,000,000 

10°  C. 

89,000 

1,940,000 

1,000,000,000 

— 

20°  C. 

4,000,000 

1,000,000,000 

~ 

Germicidal  Property.  —  Freshly  drawn  milk  appears  to  have  a 
slight  germicidal  action.  If  samples  are  examined  every  hour 
the  colonies  at  first  decrease  in  number.  Soon,  however,  this 
property  disappears  and  there  follows  a  continuous  and  sometimes 
rapid  increase.  At  temperatures  under  10°  C.  the  effect  may  be 

1  Adapted  from  Park  and  Williams,  "Pathogenic  Microorganisms,"  1917, 
p.  634. 


MILK  85 

marked  for  from  eight  to  twelve  hours ;  at  higher  temperatures  it 
is  scarcely  perceptible.  It  is  supposed  by  some  authorities  to 
be  an  agglutinative  rather  than  a  germicidal  property ;  that  is,  the 
bacteria  may  not  actually  be  destroyed  but  gathered  together 
in  clusters  that  will  not  readily  separate.  Thus  a  colony  may  result 
from  a  group  of  bacteria  instead  of  a  single  individual  and  a  wrong 
impression  of  decrease  in  number  may  be  obtained.  In  any  case 
the  action  is  comparatively  feeble  and  is  soon  lost. 

Estimation  of  Bacterial  Content.  —  The  statement  made  con- 
cerning the  bacteriological  examination  of  soil  and  water  is  equally 
applicable  to  the  examination  of  milk ;  by  no  known  method  can 
the  exact  number  present  in  a  given  sample  be  determined.  Some 
species  grow  slowly  or  not  at  all  on  culture  media;  some  are 
aerobes,  others  anaerobes ;  some  require  body  temperature,  others 
grow  best  at  a  lower  temperature.  Clusters  of  bacteria  may  re- 
main attached  even  after  vigorous  shaking  and  develop  as  one 
colony  instead  of  several.  Again,  bacteria  are  not  equally  dis- 
tributed in  the  milk  and  the  sample  may  not  be  representative. 
As  the  cream  globules  rise  they  carry  along  with  them  numbers 
of  the  bacteria  present,  until  finally  four  or  five  times  as  many 
organisms  may  be  found  in  the  cream  and  upper  layer  as  in  the 
lower  portion.  Unless  the  milk  is  thoroughly  mixed  before  the 
sample  is  taken  this  also  may  constitute  a  source  of  error. 

The  method  usually  employed  to  estimate  the  number  of  bac- 
teria present  in  a  cubic  centimeter  of  milk  is  the  plating  method 
already  described.  The  direct  microscopic  examination  of  a  film 
of  milk  has  been  advocated  in  order  to  eliminate  the  above  sources 
of  error.  A  square  centimeter  is  ruled  on  a  glass  slide  and  0.01 
c.c.  of  milk  accurately  measured  and  evenly  spread  over  it.  The 
film  is  dried  in  the  air,  fixed  with  methyl  alcohol,  the  fat  dissolved 
with  xylol,  and  finally  it  is  lightly  stained  with  methylene  blue. 
The  oil  immersion  lens  is  employed  for  the  examination,  and  the 
tube  of  the  microscope  is  so  arranged  that  the  microscopic  field 
covers  ^5-  sq.  mm.  The  average  number  of  bacteria  found  in 
each  field  is  multiplied  by  5000  to  give  the  number  of  bacteria 
contained  in  0.01  c.c.  of  milk.  When  the  results  of  the  two  methods 


86  BACTERIOLOGY  FOR  NURSES 

are  compared  it  is  generally  found  that  the  microscopic  method 
gives  a  much  higher  count  than  the  plate  method.  If,  however, 
the  clumps  of  bacteria  seen  in  the  former  are  given  only  the  value 
of  one  the  two  counts  closely  agree.  Microscopic  examination 
of  pasteurized  milk  is  not  practical,  since  it  offers  no  means  of  dis- 
tinguishing between  living  and  dead  bacteria. 

Milk  Standards.  —  Several  cities  have  endeavored  to  obtain 
a  purer  milk  supply  by  admitting  for  sale  only  the  milk  that  reaches 
the  standard  they  have  set.  The  requirements  are  based  on  farm 
conditions  and  chemical  and  bacteriological  analyses.  The  num- 
ber of  bacteria  permissible  in  the  milk  sold  in  New  York  City  is 
as  follows : 

Grade  A l  must  not  contain  more  than  60,000  bacteria  per  c.c. 
It  may  be  raw  or  pasteurized ;  the  raw  is  obtained 
from  cows  that  have  successfully  passed  the  tuber- 
culin test. 

Grade  B  is  all  pasteurized.  Before  pasteurization  it  may 
contain  1,500,000  and  after  pasteurization  50,000 
per  c.c. 

Grade  C  is  all  pasteurized.  It  may  contain  any  number  within 
reason  before  pasteurization  and  not  more  than 
100,000  per  c.c.  afterwards.  It  is  to  be  used  for 
cooking  purposes  only. 

Sour  Milk.  —  Under  ordinary  circumstances  milk,  after  a  short 
period,  becomes  sour.  The  milk  sugar,  lactose,  is  fermented  and 
lactic  acid  is  produced ;  curdling  results  as  a  precipitation  of  the 
casein  from  solution  by  the  acid. 

When  it  was  first  discovered  that  the  souring  of  milk  was  due 
to  bacteria  it  was  thought  that  only  one  species  was  responsible 
for  the  change,  and  an  organism  isolated  was  named  Bacillus 
acidi  lacti  because  it  had  the  ability  to  decompose  lactose  into 
lactic  acid.  Later  it  was  discovered  that  more  than  a  hundred 
other  species  have  the  same  power  in  a  varying  degree.  The 
amount  of  acid  produced  depends  largely  upon  the  bacteria  pro- 

1  Park  and  Williams,  "Pathogenic  Microorganisms,"  1918,  p.  642. 


MILK  87 

ducing  it.  As  soon  as  the  amount  becomes  injurious  to  the  or- 
ganism growth  ceases  and  no  more  acid  is  formed. 

Sour  milk  obtained  from  clean  milk  is  considered  beneficial  as  a 
food.  In  certain  parts  of  Asia  and  eastern  Europe  it  forms  part 
of  the  staple  diet.  Within  recent  years  similar  sour  milk  products 
have  been  manufactured  commercially  on  a  large  scale  in  western 
countries.  It  has  long  been  recognized  that  a  mutual  antagonism 
exists  between  the  acid-producing  bacteria  and  those  causing 
putrefactive  changes,  and  on  this  basis  attempts  to  combat  such 
changes  occurring  in  the  intestines,  which  lead  to  so-called  "auto- 
intoxication," were  early  made  by  adding  to  the  diet  acid-forming 
bacteria  together  with  carbohydrates.  At  first  the  results  were 
only  moderately  successful.  Then  it  occurred  to  Metchnikoff  that 
probably  the  organisms  used  had  not  the  capacity  to  produce  acid 
in  large  enough  quantities.  In  his  search  for  a  more  powerful 
acid  producer  his  attention  was  attracted  to  Bacillus  bulgaricus, 
an  organism  isolated  from  milk  in  1905  by  Massol  and  Cohendy, 
said  to  produce  as  much  as  twenty-five  grams  of  lactic  acid  per 
liter  of  milk  in  addition  to  smaller  quantities  of  other  acids.  The 
fact  that  it  does  not  attack  proteins  and  that  it  is  not  pathogenic 
makes  it  particularly  suitable  for  its  therapeutic  role. 

Putrid  Milk.  —  When  boiled  milk  is  allowed  to  stand  at  room 
temperature  it  sometimes  becomes  bitter  and  has  an  alkaline 
reaction.  A  spore-bearing  group  of  bacteria  and  also  certain  ana- 
erobes are  responsible  for  this  change ;  they  decompose  the  protein 
into  injurious  substances  resembling  "  ptomains." 

Ropy  Milk.  —  Bacteria  which  produce  this  condition  are  widely 
distributed  in  nature.  In  Europe  Bacillus  lactis  viscosus  is  con- 
sidered the  main  agent.  A  micrococcus,  two  forms  of  streptococci, 
and  certain  of  the  lactic  acid  bacilli  are  also  able  to  bring  about 
the  same  condition.  In  certain  European  countries  ropy  milk  is 
considered  a  delicacy.  It  is  not  injurious  provided  the  sliminess 
is  not  the  result  of  a  mucopurulent  discharge  from  the  udder  of 
the  cow. 

Colored  Milk.  —  A  red  color  in  milk  may  be  due  to  blood  if 
the  udder  is  diseased ;  it  may  also  appear  if  bacteria  giving  a  red 


88  BACTERIOLOGY  FOR  NURSES 

pigment,  such  as  B.  prodigiosus  or  B.  erythrogenes  be  present. 
Blue  milk  is  usually  due  to  the  presence  of  B.  cyanogenes.  Milk 
colored  by  bacteria  is  apparently  harmless. 

Pathogenic  Organisms  in  Milk.  —  There  are  two  sources  from 
which  disease-producing  bacteria  may  gain  access  to  milk :  from 
the  cow,  or  from  some  human  case.  The  latter  is  the  much  more 
common.  Bacteria  causing  typhoid  fever,  diphtheria,  or  scarlet 
fever  may  find  their  way  into  the  milk  from  carriers,  convalescents, 
or  persons  suffering  from  a  mild  form  of  the  disease  who  are  engaged 
in  handling  the  milk.  Or  infection  may  come  in  a  less  direct  way : 
contaminated  water  may  be  used  for  rinsing  milk  pails  or  flies 
may  convey  the  bacteria  from  excreta  improperly  disposed  of. 

Of  the  diseases  transmitted  to  man  from  the  cow  bovine  tuber- 
culosis and  septic  sore  throat  occur  the  most  frequently.  The 
micrococcus  causing  Malta  fever  is  usually  conveyed  by  goats' 
milk,  although  cows  are  said  to  be  susceptible  to  the  disease.  Cases 
of  foot-and-mouth  disease  transmitted  by  milk  are  extremely  rare. 

Just  how  the  bovine  tubercle  bacilli  find  their  way  into  the  milk 
supply  has  long  been  a  question  of  intense  interest.  In  the  case 
of  a  tuberculous  udder  it  is  a  simple  matter.  It  has  been  suggested 
that  cows  suffering  from  pulmonary  tuberculosis  may  cough  up 
bacilli,  swallow  them,  and  then  pass  them  in  their  feces.  As 
enormous  numbers  have  sometimes  been  found  in  feces,  and  as 
practically  all  market  milk  has  been  contaminated  with  manure 
rubbed  off  from  the  flanks  of  the  cow,  it  is  reasonable  to  assume 
that  occasionally  tubercle  bacilli  gain  access  to  the  milk  in  this 
manner. 

For  many  years  it  has  been  thought  that  bacteria  never  pass 
through  the  mammary  gland  unless  there  is  a  local  lesion.  Recent 
experiments,  however,  tend  to  prove  that  in  case  of  generalized 
tuberculosis  of  the  cow  tubercle  bacilli  may  pass  into  the  milk 
without  any  evidence  of  the  udder  being  diseased. 

To  what  extent  milk  contaminated  with  bovine  tubercle  bacilli 
is  responsible  for  human  tuberculosis  is  still  an  undecided  question. 
It  is  stated  that  in  districts  where  the  milk  from  tuberculous  cows 
is  consumed  the  children  are  frequently  found  to  suffer  from 


MILK  89 

diseases  of  the  joints  and  cervical  glands  and  that  tubercle  bacilli 
of  the  bovine  type  have  been  obtained  from  these  lesions.  This 
may  be  explained  by  the  fact  that  children  drink  more  milk  than 
adults  and  that  they  are  more  susceptible  to  the  bovine  type. 
Certain  authorities  hold  the  view  that  pulmonary  tuberculosis 
in  adults  may  be  accounted  for  by  an  infection  contracted  in  child- 
hood due  to  milk.  That,  however,  is  by  no  means  a  general  opinion. 

The  mode  of  access  of  the  tubercle  bacilli  from  human  cases 
of  the  disease  is  quite  easily  conceived.  A  milker  suffering  from 
tuberculosis  of  the  lungs,  whose  fingers  have  come  in  contact  with 
his  sputum,  might  readily  wash  off  enormous  numbers  of  bacilli 
into  the  milk  pail,  or  droplets  expelled  from  the  mouth  while  talk- 
ing or  coughing  might  carry  their  quota. 

Septic  Sore  Throat.  —  Many  epidemics  of  septic  sore  throat 
have  occurred  directly  traceable  to  the  milk  supply,  and  several 
varieties  of  streptococci  have  been  isolated  as  the  causal  agents. 
It  is  assumed  that  the  majority  of  outbreaks  are  due  to  organ- 
isms derived  indirectly  from  human  sources.  The  streptococci 
which  produce  garget  in  cows  do  not  produce  sore  throats  in  human 
beings,  and  conversely,  the  species  of  streptococci  which  produce 
tonsillitis  in  man  are  only  slightly  pathogenic  for  cows.  Experi- 
ments have  shown  that  the  streptococci  giving  rise  to  septic  sore 
throat  may  find  their  way  into  the  milk  ducts  when  the  teats 
have  been  wiped  with  an  infected  cloth ;  thus  they  may  become 
implanted  in  the  udder  of  the  cow  and  continue  to  grow  for  several 
weeks  without  giving  rise  to  any  form  of  disease.  In  this  way 
the  cow  may  be  a  "  carrier  "  of  the  human  variety  of  strepto- 
cocci. 

Foot  and  Mouth  Disease  has  been  reported  to  have  been  trans- 
mitted to  man  through  dairy  products  coming  from  diseased  cows. 
In  many  the  disease  is  very  mild  and  seldom  fatal. 

Infantile  Diarrhea.  —  Whether  the  majority  of  cases  of  infantile 
diarrhea  are  due  to  the  bacterial  content  of  dirty,  stale  milk  or 
to  its  changed  chemical  content  is  not  yet  decided.  While  no 
single  organism  has  been  isolated  that  can  truly  be  said  to  produce 
the  malady,  it  remains  true  that  in  localities  where  fresh,  clean 


90  BACTERIOLOGY  FOR  NURSES  j 

milk  is  supplied  cases  of  infantile  diarrhea  are  of  much  less  fre- 
quent occurrence. 

Typhoid  Fever.  —  The  typhoid  bacillus  is  the  cause  of  more 
milk-borne  epidemics  than  any  other  organism ;  yet  it  is  seldom 
that  it  can  be  isolated.  Generally,  however,  examination  of  the 
feces  of  the  employees  handling  the  milk  reveals  a  convalescent 
case  or  a  "  carrier."  In  some  cases  infection  has  been  traced  to 
the  use  of  contaminated  water  for  washing  the  milk  utensils.  In 
Washington  during  the  four  years  1907-1910  10  per  cent  of  all 
the  cases  of  typhoid  fever  were  traced  to  milk.1 

Scarlet  Fever.  —  Although  the  organism  causing  scarlet  fever 
is  as  yet  unknown  many  epidemics  have  occurred,  presumably 
due  to  milk  infected  from  human  sources. 

Diphtheria.  —  Milk-borne  epidemics  of  diphtheria  are  less  fre- 
quent than  those  of  typhoid  or  scarlet  fever.  The  source  is 
generally  a  convalescent  case  or  a  carrier. 

General  Character  of  Milk-borne  Epidemics.  —  An  explosive 
onset  and  a  gradual  decline  generally  indicate  a  contaminated 
milk  or  water  supply.  If  the  bacteria  in  the  milk  are  compara- 
tively few  the  disease  may  only  appear  in  a  few  susceptible  persons 
who  drink  it;  if  the  organisms  are  numerous  the  infection  may 
be  carried  along  the  entire  milk  route.  At  first  the  disease  appears 
only  amongst  those  who  have  partaken  of  the  infected  milk ;  later, 
secondary  cases  may  appear. 

Sterilization  of  Milk.  —  With  the  realization  of  the  possibility 
of  disease-producing  organisms  being  present  in  milk  the  question 
arises  as  to  the  best  method  of  destroying  them  and  rendering 
the  milk  safe  as  a  food.  Sterilization  by  heat  is  the  only  practical 
method.  Boiling,  however,  is  objected  to  by  pediatricians  on  the 
ground  that  cases  of  scurvy  and  rickets  are  likely  to  develop  in 
infants  fed  exclusively  on  boiled  milk.  Fortunately  these  dangers 
can  be  obviated  if  instead  of  being  boiled  the  milk  is  pasteurized. 

Pasteurization.  —  The  process  devised  by  Pasteur  for  preserv- 
ing wines  without  loss  of  their  original  flavor  is  found  to  be  equally 
well  adapted  for  the  treatment  of  milk.  As  the  object  of  pasteur- 
1  Rosenau,  "Preventive  Medicine  and  Hygiene,"  p.  574. 


MILK  91 

ization  is  the  destruction  of  the  pathogenic  organisms  without 
so  changing  the  food  constituents  that  it  is  less  suitable  for  infant 
feeding,  the  temperature  used  and  the  length  of  time  of  exposure 
depend  on  these  two  points.  The  lowest  temperature,  therefore, 
that  will  kill  non-spore-bearing  bacteria  in  a  reasonable  length  of 
time  is  the  one  chosen.  An  exposure  to  60°  C.  for  twenty  minutes 
or  to  70°  C.  for  five  minutes  has  been  found  to  be  efficient.  It  is 
advisable  in  commercial  practice  where  milk  is  pasteurized  in  large 
quantities  to  increase  the  temperature  a  few  degrees  and  prolong 
the  heating  ten  to  fifteen  minutes. 

There  are  three  methods  in  general  use : 

I.  The  flash  method  consists  in  heating  the  milk  to  81°  C.  and 
chilling  it  at  once.  It  is  the  quickest  and  cheapest 
method,  but  the  least  reliable. 

II.  The  holding  method  consists  in  heating  the  milk  to  65°  C., 
then  holding  it  at  that  temperature  from  thirty  to  forty- 
five  minutes.  Specially  devised  tanks  have  been  con- 
structed as  "  holders  "  that  give  excellent  results.  The 
method  has  proved  most  satisfactory  for  commercial 
purposes. 

III.  Pasteurization  in  the  bottle  is  the  ideal  method.  All  danger 
of  recontamination  is  thus  eliminated.  The  bottles  are 
tightly  stoppered,  immersed  in  a  water  bath,  brought  to 
the  required  temperature,  and  held  there  a  sufficient  length 
of  time. 

Whichever  method  of  pasteurization  is  employed  rapid  cooling  is 
of  great  importance. 

The  following  experiment  well  illustrates  the  result  of  pasteuri- 
zation so  far  as  diminution  in  the  number  of  bacteria  is  concerned.1 
The  milk  was  heated  to  70°  C.  for  a  half  and  for  one  minute. 

SAMPLE  I 

Raw  milk 600,000  bacteria  per  o.c. 

|  minute  pasteurization 2,000  bacteria  per  c.c. 

1  minute  pasteurization 1,000  bacteria  per  c.c. 

1  Adapted  from  Park  and  Williams,  "  Pathogenic  Microorganisms,"  p.  635. 


92 


BACTERIOLOGY  FOR  NURSES 


SAMPLE  II 

Raw  milk 5,400,000  bacteria  per  c.c. 

J  minute  pasteurization      ....  7,400  bacteria  per  c.c. 

1  minute  pasteurization      ....  600  bacteria  per  c.c. 

The  pasteurization  of  dirty  milk  containing  many  millions  of 
bacteria  is  a  rather  questionable  procedure.  The  number  of 
microorganisms  may  be  considerably  reduced,  but  all  deleterious 
qualities  will  not  be  entirely  removed. 

A  striking  illustration  of  the  beneficial  effect  of  the  pasteuriza- 
tion of  milk  of  an  average  quality  is  given  below. 

When  the  children  in  a  New  York  institution  (Randall's  Island) 
were  given  milk  from  a  selected  herd  pastured  on  the  island  the 
death  rate  was  as  follows : 1 


CHILDREN 
TREATED 

NUMBER  OP 
DEATHS 

PERCENTAGE 

During  the  years  1895  to  1897  inclusive 

3,609 

1,509 

48.81 

A  pasteurizing  plant  was  installed  in  the  early  part  of  1898.     No  other 
change  in  diet  or  hygiene  was  made. 


CHILDREN 
TREATED 

NUMBER  OF 
DEATHS 

PERCENTAGE 

During  the  years  1898  to  1904  inclusive 

6,200 

1,349 

21.75 

Butter.  —  Cream  from  which  butter  is  to  be  made  is  usually  al- 
lowed to  sour  or  "  ripen  "  ;  that  is,  it  is  purposely  allowed  to  stand 
in  a  container  two  or  three  days  in  order  that  the  bacteria  present 
may  develop  a  characteristic  flavor  and  aroma.  The  process  is 
one  of  decomposition,  but  up  to  a  certain  point  it  gives  pleasurable 
and  profitable  results;  beyond  that  point  it  is  undesirable  and 
offensive.  Ordinarily  it  is  stopped  at  the  right  moment.  The 
method  usually  employed  is  that  of  allowing  the  cream  to  ripen 
under  the  influence  of  any  species  of  bacteria  that  happen  to  be 
present.  Occasionally,  however,  by  this  "  hit  or  miss  "  procedure 

1  Adapted  from  Jordan,  "General  Bacteriology,"  1917,  p.  561. 


MILK  93 

organisms  that  produce  disagreeable  flavors  gain  ascendancy  and 
the  subsequent  butter  is  of  a  low  grade. 

In  some  dairies  it  is  the  custom  to  use  a  "  natural  starter  " ; 
that  is,  a  small  quantity  of  cream  that  has  developed  the  required 
qualities  is  added  to  the  fresh  cream.  This  procedure  is  simply 
seeding  the  new  cream  with  the  organisms  known  to  be  capable 
of  producing  the  desired  results. 

A  step  further  has  been  taken  by  the  more  progressive  dairy- 
men. The  fresh  cream  is  pasteurized  in  order  to  eliminate  the 
action  of  any  bacteria  present  and  a  pure  culture  of  an  organism 
already  proved  to  have  the  requisite  qualities  is  introduced.  In 
this  way  butter  of  a  uniform  quality,  at  least  as  regards  flavor, 
can  always  be  depended  on. 

Unfortunately  all  cream  is  not  pasteurized  before  it  is  churned, 
and  pathogenic  organisms  are  by  no  means  rare  in  market  butter. 
In  an  examination  of  21  samples  offered  for  sale  in  Boston  two  or 
9.5  per  cent  were  found  to  contain  tubercle  bacilli.1  Other  investi- 
gations in  another  locality  report  15.2  per  cent.  Many  experi- 
ments have  been  made  to  determine  the  length  of  time  typhoid 
bacilli  will  continue  to  live  in  butter.  It  is  generally  assumed 
that  they  die  after  a  few  days. 

Cheese.  —  The  bacteriology  of  cheese  making  is  somewhat 
more  indefinite  than  that  of  butter  making.  It  is  proven  that 
cheeses  are  ripened  by  the  action  of  bacteria  and  molds  and  that 
the  different  flavors  are  due  to  the  growth  of  different  species  dur- 
ing the  process.  As  yet,  however,  the  organisms  concerned  and 
the  role  they  play  is  largely  a  matter  of  conjecture. 

1  Rosenau,  "Preventive  Medicine,"  p.  581. 


PART  II 


CHAPTER   IX 


Infection.  —  In  the  early  days  of  bacteriology  the  presence  of 
bacteria  in  or  on  the  skin  or  mucous  membranes  was  regarded  as 
an  evidence  of  a  diseased  condition.  It  is  well  known  now,  how- 
ever, that  organisms  such  as  streptococci,  staphylococci,  and 
pneumococci  are  frequently  present  in  the  nose  or  mouth  or  on 
the  skin  of  normal  healthy  persons.  The  intestines  contain  many 
thousands  of  different  species,  but  only  under  unusual  conditions 
do  they  produce  disease.  The  mere  contact,  therefore,  of  micro- 
organisms with  bodies  of  animals  or  man  does  not  necessarily 
mean  a  diseased  condition.  When,  however,  they  pass  the  pro- 
tective skin  and  membranes,  invade  the  deeper  tissues,  and  multiply 
there  they  may  produce  poisons  which  give  rise  to  the  various 
symptoms  met  with  in  disease.  This  invasion,  multiplication,  and 
resulting  disease  is  spoken  of  as  an  infection. 

In  the  production  of  an  infection  the  main  factors  are  (1)  the 
defensive  forces  the  body  can  command  to  resist  the  invaders 
and  (2)  the  power  of  the  invading  organism  to  withstand  all  the 
opposing  forces  the  body  can  produce  against  it,  to  multiply  and 
to  elaborate  poisonous  substances. 

Certain  bacteria  may  live  and  multiply  in  the  body  apparently 
without  either  causing  or  receiving  injury.  Such  organisms  may 
be  harmless  and  incapable  of  producing  poisons  or  there  may  be 
established  between  them  and  the  body  cells  an  equilibrium  in 
that  the  amount  of  poison  produced  is  neutralized  and  rendered 
inert  by  a  corresponding  amount  of  cell  secretions.  In  the  latter 

94 


ABILITY  OF  BACTERIA  TO  PRODUCE  DISEASE    95 

case  if  the  invaders  retain  their  pathogenic  powers  the  person 
thus  harboring  them  and  probably  disseminating  them  is  termed 
a  germ  "  carrier."  Thus  pneumococci  and  influenza  bacilli 
are  present  in  the  nose  or  throat  of  many  individuals.  Persons 
who  have  recovered  from  an  attack  of  diphtheria  or  who  have  been 
in  contact  with  those  suffering  from  the  disease  frequently  become 
carriers.  The  typhoid  bacillus  may  remain  located  in  the  gall 
bladder  and  be  discharged  in  the  feces  long  after  all  symptoms  of 
the  disease  have  disappeared.  It  has  been  estimated  that  after 
convalescence  from  typhoid  fever  one  to  three  per  cent  may  remain 
carriers  for  months  or  years. 

When  the  balance  of  such  a  relationship  is  disturbed  by  dimin- 
ished resistance  on  the  part  of  the  body  infection  is  likely  to  occur. 
Such  an  infection  is  spoken  of  as  autogenous.  Appendicitis  result- 
ing from  infection  by  the  colon  bacillus  following  congestion  due 
to  fecal  impaction  may  be  taken  as  an  example.  Usually,  however, 
infections  result  from  contact  with  contaminated  material  outside 
of  the  body.  Probably  those  conveyed  by  water  or  food  are  of  the 
most  frequent  occurrence ;  for  example,  typhoid  bacilli  or  cholera 
spirilla  by  water,  tubercle  bacilli  by  milk.  Or  infection  may  result 
from  a  scratch  with  a  rusty  nail  on  which  are  the  spores  of  tetanus, 
or  hydrophobia  from  the  bite  of  a  rabid  dog. 

Infections  resulting  from  the  introduction  of  bacteria  from 
sources  apart  from  the  individual  infected  are  spoken  of  as  ex- 
ogenous. 

Contagious  and  Infectious  Diseases.  —  Organisms  that  are 
strictly  parasitic  and  therefore  cannot  grow  apart  from  the  human 
body  must,  in  order  to  produce  disease  in  a  second  person,  be 
transferred  from  one  person  to  another  by  direct  contact;  the 
leprosy  bacillus  may  be  taken  as  an  example.  Diseases  produced 
by  these  organisms  are  said  to  be  contagious.  Other  organisms 
not  so  strictly  parasitic,  which  are  able  to  adapt  themselves  to 
other  conditions  outside  of  the  body,  may  gain  access  to  a  second 
individual  by  means  of  contaminated  material.  A  disease  thus 
produced  is  spoken  of  as  infectious.  No  strict  rule,  however, 
can  be  adhered  to  in  such  a  classification  since  bacteria  commonly 


96  BACTERIOLOGY   FOR  NURSES 

transmitted  by  the  latter  method  may  under  certain  conditions 
be  transmitted  by  the  former.  A  simpler  plan  is  the  classification 
of  all  bacterial  diseases  as  infectious,  reserving  the  term  contagious 
only  for  those  which  are  contracted  as  a  result  of  direct  contact. 

Defensive  Forces  of  the  Body.  —  The  body  possesses  three 
natural  defenses  against  bacterial  invasion :  (1)  a  covering  more  or 
less  unsuitable  for  bacterial  growth  and  penetration;  (2)  the 
ability  to  produce  chemical  substances  which  either  kill  the  organ- 
isms or  render  their  poisons  inert ;  (3)  the  power  of  certain  cells  to 
engulf  and  destroy  the  invaders.  Thus  even  though  the  first 
barrier  be  passed  invasion  does  not  necessarily  mean  infection; 
the  other  forces  acting  singly  or  together  may  speedily  prevent 
injury. 

Influence  of  Tissues  on  Bacterial  Invasion.  —  Many  species  of 
bacteria  find  a  temporary  lodgment  upon  the  skin,  but  it  is  a  poor 
soil  for  growth  and  forms  an  effective  barrier  against  entrance 
into  the  deeper  tissues.  A  group  of  cocci,  however,  are  habitually 
present,  and  injuries  such  as  wounds,  or  burns,  or  sometimes  a 
simple  pin  prick  enable  them  to  penetrate  deeper.  Certain  varie- 
ties are  able  to  produce  direct  action  without  the  existence  of 
previous  injury.  Thus  staphylococci  may  reach  the  roots  of  hair 
follicles  and  sweat  glands  and  cause  suppurative  conditions. 

The  warmth  and  moisture  of  the  mucous  membranes  make 
them  much  better  adapted  for  bacterial  growth  than  the  skin. 
The  nasal  cavity  is  somewhat  cleansed  by  the  nasal  secretion. 
Nevertheless,  the  influenza  bacillus,  the  diphtheria  bacillus,  and 
others  find  it  possible  to  obtain  a  lodgment  and  by  concentrating 
at  one  point  to  lower  the  vitality,  destroy  the  epithelial  tissue,  and 
gain  an  entrance  there.  It  is  possible  that  the  meningococcus 
and  the  virus  of  anterior  poliomyelitis  gain  access  to  the  body  by 
this  route. 

Diminution  in  quantity  or  change  in  quality  of  the  normal 
body  secretions  may  favor  the  growth  of  bacteria  and  render  in- 
vasion easier.  For  example,  the  saliva  is  ordinarily  somewhat 
bactericidal,  but  during  a  fever  the  amount  secreted  is  diminished 
and  unless  the  mouth  is  carefully  and  frequently  cleansed  fetid 


ABILITY  OF  BACTERIA  TO  PRODUCE  DISEASE    97 

sores  develop  on  the  teeth  and  lips  as  a  result  of  bacterial 
growth. 

The  gastric  juice  through  the  hydrochloric  acid  it  contains  has 
a  marked  germicidal  effect.  Nevertheless,  many  bacteria  escape 
its  action  because  they  are  protected  in  the  food  or  because  of  its 
neutralization.  Bacteria  causing  typhoid  fever,  cholera,  tuber- 
culosis, and  other  infections  may  thus  pass  unharmed  into  the 
intestines  and  there  produce  their  respective  lesions. 

Most  inhaled  organisms  which  pass  the  larynx  are  gradually  re- 
moved by  the  ciliated  epithelium  of  the  bronchi ;  the  few  which 
succeed  in  gaining  entrance  to  the  lungs  are  able  to  multiply  and 
produce  disease  only  when  the  lung  tissues  have  lost  some  of  their 
resistant  powers. 

Points  of  Entrance.  —  Infection  occurs  with  certain  bacteria 
only  when  they  enter  the  body  by  an  appropriate  route  and  reach 
special  tissues.  Thus  typhoid,  cholera,  and  dysentery  infection 
does  not  take  place  unless  the  organisms  enter  the  gastro-intestinal 
tract ;  they  never  enter  through  the  skin.  Gonococci  usually  enter 
the  body  through  the  genital  organs  or  occasionally  the  eye,  but 
never  by  way  of  the  respiratory  or  digestive  tract.  The  avenue 
of  invasion  thus  determines  largely  whether  infection  will  or  will 
not  occur,  and  if  it  does  its  nature  and  severity.  Diphtheria 
bacilli  rubbed  on  an  abrasion  of  the  hand  produces  only  a  slight 
lesion,  but  rubbed  on  an  abrasion  of  the  throat  they  cause  inflamma- 
tion, necrosis  of  the  tissue,  and  a  general  poisoning.  Pneumococci 
lodging  on  the  surface  of  the  eye  may  cause  a  severe  conjunctivitis, 
on  the  mucous  membrane  of  the  throat  a  pseudomembranous 
angina,  and  in  the  lungs  pneumonia. 

Several  species  of  bacteria  are  normally  present  in  the  mouth, 
some  of  which  may  be  the  cause  of  caries  of  the  teeth.  The  im- 
portance of  this  condition  is  becoming  more  and  more  recognized 
as  having  a  direct  bearing  on  the  general  health.  A  carious  tooth 
may  be  the  portal  of  entrance  for  microorganisms  causing  a  gen- 
eral infection. 

The  mode  of  entrance  of  the  tubercle  bacilli  is  still  a  disputed 
point.  The  theory  that  they  enter  through  the  respiratory  tract 


98  BACTERIOLOGY  FOR  NURSES 

accounts  most  readily  for  the  far  greater  frequency  with  which 
tuberculosis  affects  the  lungs  than  it  does  other  parts  of  the  body. 
Recent  experiments,  however,  tend  to  show  that  they  may  be 
swallowed  and  pass  through  a  practically  intact  intestinal  wall 
and  find  their  way  into  the  mesenteric  lymph  glands.  The  cer- 
vical glands  of  children  often  become  infected  with  tubercle 
bacilli  without  any  visible  trace  being  left  in  the  mucous  membrane 
of  the  larynx  to  indicate  their  passage.  In  such  cases  the  crypts 
of  the  tonsils  are  strongly  suspected  of  being  their  portal  of  en- 
trance. 

Bones  are  infected  by  organisms  carried  to  them  in  the  blood 
stream  except  in  cases  of  direct  injury ;  for  this  reason  the  perios- 
teum and  the  bone  marrow,  being  most  abundantly  supplied  with 
blood,  are  first  affected. 

Influence  of  Numbers.  —  The  number  of  pathogenic  bacteria 
which  succeed  in  invading  the  tissues  is  an  important  factor  in 
determining  whether  or  not  an  infection  will  take  place.  If  only 
a  few  gain  entrance  all  may  be  killed ;  if,  however,  a  large  number 
are  introduced  some  are  almost  sure  to  survive,  to  multiply,  and 
produce  their  specific  disease  unless  the  body  is  immune.  When  a 
chronic  disease  or  other  factors  have  reduced  the  body  defenses 
fewer  bacteria  than  would  otherwise  be  required  may  give  rise  to 
infection. 

Virulence.  —  The  degree  of  ability  which  an  organism  possesses 
to  overcome  the  defensive  forces  of  the  body  and  to  give  rise  to 
disease  is  spoken  of  as  its  virulence.  Bacteria  whose  virulence 
is  great  may  produce  disease  when  only  few  in  number,  whereas 
millions  of  a  less  virulent  species  might  be  required. 

It  is  impossible,  by  any  means,  to  make  a  known  non-virulent 
organism  virulent.  It  is  not  difficult,  on  the  other  hand,  to  decrease 
or  increase  the  virulence  of  an  organism  already  possessing  patho- 
genic powers.  The  ability  to  produce  poison  may  be  lessened 
by  repeated  growth  on  artificial  culture  media ;  by  exposure  of  a 
culture  for  a  short  period  to  a  temperature  just  below  the  thermal 
death  point,  or  to  sunlight,  or  to  small  quantities  of  antiseptic 
or  germicidal  substances.  These  methods  are  frequently  employed 


ABILITY  OF  BACTERIA  TO  PRODUCE  DISEASE    99 

to  attenuate  cultures  in  the  preparation  of  vaccines  to  be  used 
for  purposes  of  active  immunization. 

Ordinarily,  the  passage  of  an  organism  through  an  animal 
increases  its  pathogenicity  only  for  that  particular  species  of 
animal.  Thus  the  passage  of  certain  bacteria  through  a  guinea 
pig  increases  their  virulence  for  guinea  pigs  and  not  for  rabbits 
nor  rats.  A  method  of  increasing  the  virulence  of  a  given  culture 
is  to  inclose  it  in  a  collodion  capsule  of  suitable  thickness  and 
place  the  capsule  within  the  abdominal  cavity  of  the  chosen  ani- 
mal. The  body  fluids  are  able  to  transfuse  through  the  sac,  de- 
stroying such  bacteria  as  are  unable  to  withstand  their  injurious 
influence.  In  this  way  only  the  strongest  survive  and  a  race 
of  more  virulent  organisms  results. 

An  exception  occurs  in  the  passage  of  the  smallpox  virus  through 
the  calf,  where  it  loses  forever  its  power  of  producing  smallpox. 

Certain  bacteria  increase  their  resistance  against  the  body  defen- 
sive forces,  and  consequently  their  virulence  by  the  formation  of 
a  capsule.  The  capsule  may  be  quickly  lost  when  the  organism 
is  grown  on  artificial  culture  medium,  and  its  virulence  correspond- 
ingly lowered.  Repeated  passage  through  animals  will  again  re- 
store it  and  produce  a  race  of  capsulated  virulent  bacteria. 

It  is  thought  by  some  authorities  that  bacteria  actively  secrete 
substances  that  are  able  to  paralyze  the  protective  forces  of  the 
body,  especially  the  leucocytes.  Little  is  known  of  them  or  their 
action.  Their  existence,  nevertheless,  may  explain  the  statement 
made  by  Metchnikoff,  that  a  virulent  microorganism  is  not  so 
readily  taken  up  by  the  leucocytes  as  a  non-virulent  one.  Thus 
it  would  seem  that  in  an  infection  a  tremendous  struggle  is  carried 
on  between  the  bacteria  and  the  body  cells,  each  side  provoked 
by  the  other  to  manufacture  forces  that  will  either  attack  the 
enemy  or  protect  itself  against  counter  attacks. 

Mixed  and  Secondary  Infections.  —  Several  different  microor- 
ganisms may  invade  the  tissues  at  the  same  time  and  produce  a 
mixed  infection,  or  one  may  follow  another  or  others  and  give  rise 
to  secondary  infection.  Associated  organisms  are  often  influenced 
by  the  activities  of  each  other.  Thus  the  presence  of  pus-produc- 


100  BACTERIOLOGY   FOR  NURSES 

ing  cocci  has  an  injurious  effect  on  anthrax  bacilli.  On  the  other 
hand,  aerobic  bacilli  make  possible  the  growth  of  anaerobes  by  ab- 
sorbing free  oxygen.  Tetanus  bacilli  and  their  spores  would  be 
less  likely  to  develop  in  wounds  were  it  not  for  the  presence  of 
aerobic  bacteria  introduced  with  them.  Blood  infections  are  us- 
ually due  to  one  form  of  bacteria  only.  Even  when  several  varie- 
ties are  introduced  only  one  as  a  rule  survives  and  multiplies.  It 
has  been  stated  that  the  presence  of  one  organism  may  increase 
the  virulence  of  another;  for  example,  the  scarlet  fever  virus  is 
said  to  favor  the  development  of  streptococci.  It  is  rather  more 
probable  that  the  factor  favoring  the  growth  of  the  latter  organisms 
is  the  reduced  resistance  of  the  tissues  due  to  the  poison  produced 
by  the  former.  On  the  other  hand,  the  products  of  certain  bacteria 
may  rid  the  body  of  certain  other  forms.  Pasteur  was  able  with 
attenuated  chicken  cholera  cultures  to  produce  immunity  against 
anthrax.  The  ingestion  of  soured  milk  with  its  enormous  numbers 
of  lactic  acid  bacteria  is  advocated  in  order  that  a  harmless  variety 
may  crowd  out  in  the  intestines  more  dangerous  organisms. 

Pathogenic  Effects  Produced  by  Bacteria.  —  As  already  stated, 
bacteria  may  be  roughly  divided  into  two  classes:  saprophytes 
and  parasites.  No  strict  dividing  line  can  be  drawn  since  many 
species  may  enter  one  or  the  other  class  according  to  conditions. 
A  similar  statement  applies  to  the  terms  pathogenic  and  non-patho- 
genic. No  known  organism  will  under  all  circumstances  produce 
disease  in  all  animals ;  conversely,  ordinary  saprophytes  may  de- 
velop both  parasitic  and  pathogenic  powers  when  the  body  resist- 
ance is  sufficiently  reduced  by  fatigue,  exposure,  or  another  infec- 
tion. Again  an  organism  that  is  highly  pathogenic  for  one  animal 
may  be  quite  harmless  for  another.  The  terms  then  are  relative. 
A  microorganism  is  pathogenic  only  when  the  defenses  of  the  body 
are  not  strong  enough  to  resist  it. 

Bacteria  may  be  termed  pathogenic  when  they  produce  one 
or  more  of  the  following  conditions:  (1)  mechanical  injury  to  the 
tissues;  (2)  disintegration  of  tissues  to  furnish  themselves  with 
food,  or  (3)  irritation  and  destruction  of  tissues  by  poisons. 

Bacteria  possessing,  great  vitality  may,  as  already  stated,  pass 


ABILITY  OF  BACTERIA  TO  PRODUCE  DISEASE     101 

into  the  deeper  tissues  and  through  the  lymphatics  gain  access  to 
the  blood  stream,  thus  giving  rise  to  bacteremia  or  septicemia. 
When  organisms  are  present  there  in  great  numbers  or  are  bunched 
together  or  mixed  with  fibrin  to  cause  thrombi  and  later  emboli, 
they  may  by  thus  blocking  the  circulation  cause  serious  mechanical 
injury.  Emboli  stationed  in  the  capillaries  of  feebly  resistant 
tissues  usually  result  in  local  lesions ;  multiple  abscesses  may  thus 
be  formed,  producing  the  condition  known  as  pyemia.  It  will 
be  noted  that  the  term  septicemia  is  applied  to  conditions  in  which 
bacteria  circulate  and  multiply  within  the  blood,  giving  rise  to 
symptoms  of  general  poisoning,  without,  however,  the  formation 
of  abscesses.  In  pyemia,  on  the  other  hand,  abscesses  are  produced 
in  the  internal  organs  and  other  parts  of  the  body. 

The  tissue  changes  produced  by  bacteria  are  either  of  a  degen- 
erative or  a  recreative  nature.  In  the  former  resistance  is  lacking 
and  the  tissue  is  finally  disintegrated ;  in  the  latter  case  there  is 
excessive  activity  on  the  part  of  the  body  cells,  secretions  are 
increased,  phagocytosis  is  marked,  until  finally  the  invaders  are 
either  eliminated  or  gain  the  battle. 

A  local  inflammatory  reaction  presents  different  characters  in 
different  conditions.  It  may  be  accompanied  by  an  exudate  serous, 
fibrinous,  or  purulent  in  character ;  it  may  be  localized  or  show  a 
tendency  to  spread ;  it  may  be  followed  by  suppuration  and  lead 
to  necroses.  In  many  diseases  the  reaction  is  somewhat  protracted 
and  there  is  a  tendency  to  the  formation  of  new  tissue.  In  leprosy, 
tuberculosis,  syphilis,  etc.,  such  formation  frequently  occurs  in 
separate  foci  so  that  nodules  result. 

Changes  unassociated  with  the  presence  of  bacteria  may  occur 
in  certain  organs,  due  to  the  action  of  bacterial  poisons  circulating 
in  the  blood;  secreting  cells  and  the  walls  of  blood  vessels  may 
thus  be  permanently  injured.  Diphtheria  poison  produces  marked 
degenerative  changes  both  in  the  spinal  cord  and  in  the  peripheral 
nerves.  It  is  possible  that  some  of  the  lesions  of  the  nervous 
system  occurring  in  syphilis  are  due  to  toxin. 

Acute  infections  ordinarily  pass  through  the  following  stages. 
First  occurs  the  period  of  incubation,  which  begins  at  the  time  of 


102  BACTERIOLOGY  FOR  NURSES 

infection  and  continues  until  the  first  symptoms  appear;  the 
period  is  more  or  less  constant  for  each  species  of  microorganism. 
According  to  the  theory  of  Vaughan  the  bacteria  are  at  this  time 
multiplying  enormously  and  for  their  food  are  using  the  proteins 
of  the  body.  Thus  the  soluble  proteins  of  the  blood  and  lymph  are 
being  converted  into  bacterial  cells.  The  process  is  entirely  one 
of  construction ;  no  bacterial  poisons  are  being  eliminated  to  dis- 
turb the  body  and  consequently  no  symptoms  appear.  The 
period  of  incubation  is  nevertheless  critical  since  a  strongly  virulent 
organism  will  develop  with  great  rapidity  and  consequently  con- 
sume a  large  amount  of  protein. 

The  second  state  is  marked  by  the  appearance  of  symptoms. 
The  body  cells  have  become  aware  of  the  presence  of  the  invaders 
and  are  now  pouring  out  ferments  they  have  generated,  which  kill 
many  of  the  bacteria  and  decompose  their  protoplasm  into  simpler 
substances,  some  of  which  are  poisonous  and  produce  the  general 
symptoms  of  intoxication. 

Then  follows  a  period  of  high  fever  and  the  disease  is  at  its  height. 
Special  symptoms  appear  according  to  the  organs  involved.  The 
struggle  between  the  invasive  forces  of  the  parasite  and  the  defen- 
sive forces  of  the  body  nears  a  climax ;  supremacy  seems  to  be  first 
with  one  side,  then  with  the  other.  Finally  the  battle  ends,  and 
if  the  body  cells  have  proved  victorious  the  symptoms  begin  to 
disappear  and  a  period  of  convalescence  commences,  during  which 
the  body  gradually  overcomes  the  effects  of  the  bacterial  invasion 
and  returns  to  a  state  of  health.  The  latter  period  is  decidedly 
critical ;  undue  exertion  or  errors  in  diet  may  further  weaken  the 
already  exhausted  tissues,  thus  lowering  their  resistance  and  lead- 
ing to  a  relapse. 

Degrees  of  Infection.  —  When  an  infection  occurs  with  a  partic- 
ularly virulent  microorganism  there  may  be  so  much  poison  liber- 
ated that  the  cells  of  the  body  are  paralyzed  and  completely  over- 
come. In  such  a  case  instead  of  a  high  fever  being  produced  the 
temperature  drops  to  subnormal,  and  rapid  prostration  and  death 
of  the  patient  quickly  ensue.  Such  an  infection  is  termed  a  malig- 
nant infection. 


ABILITY  OF  BACTERIA  TO  PRODUCE  DISEASE     103 

The  type  of  infection  already  described,  with  its  period  of  incuba- 
tion, symptoms,  high  fever,  decline,  and  convalescence,  is  spoken 
of  as  an  acute  infection. 

A  third  type  characterized  by  a  slow  development  and  mild 
symptoms  and  terminating  after  months  or  years  either  in  death 
or  recovery  is  spoken  of  as  a  chronic  infection.  In  these  infections 
it  may  be  that  the  parasites  develop  and  produce  their  toxins  very 
slowly  or  that  they  are  only  partially  absorbed  by  the  tissues.  In 
this  way  the  body  cells  are  only  stimulated  to  produce  sufficient 
protective  substances  for  their  immediate  need.  Gradually, 
however,  the  body  cells  may  become  exhausted  and  unless  aroused 
by  some  such  means  as  the  administration  of  a  vaccine  to  produce 
an  oversupply  of  antibodies  they  may  be  slowly  but  surely  over- 
come. 

The  Spread  of  Infection.  —  The  amount  of  infectious  material 
and  the  path  by  which  it  is  discharged  play  perhaps  the  largest 
role  in  the  dissemination  of  disease.  In  diphtheria,  typhoid  fever, 
cholera,  influenza,  gonorrhea,  and  pulmonary  tuberculosis  enormous 
numbers  of  virulent  bacteria  leave  the  body  in  the  discharges 
from  the  mouth,  nose,  intestines,  or  genito-urinary  tract.  Con- 
versely, in  such  diseases  as  streptococcic  meningitis,  gonorrhea! 
rheumatism,  and  tuberculous  peritonitis  there  is  little  danger  of 
infecting  others  because  few  or  no  living  bacteria  are  'discharged 
from  the  body. 

The  species  of  animals  that  may  be  infected  has  a  slight  influence 
in  the  spread  of  disease.  Anthrax,  glanders,  tuberculosis,  hydro- 
phobia, and  some  other  diseases  appear  both  in  man  and  animals ; 
certain  other  infections  such  as  gonorrhea,  syphilis,  measles, 
typhoid  fever,  etc.,  never,  so  far  as  is  known,  occur  in  animals, 
and  consequently  are  not  transmitted  by  them. 

Another  important  factor  is  the  resistance  of  the  specific  organ- 
ism to  conditions  outside  of  the  body.  Spore-bearing  bacteria, 
such  as  anthrax  and  tetanus  bacilli,  will  retain  their  pathogenic 
powers  for  years.  Certain  non-spore-forming  organisms,  such  as 
those  causing  influenza,  gonorrhea,  and  syphilis,  are  extremely 
sensitive ;  pneumococci  and  cholera  spirilla  are  a  little  more  resist- 


104  BACTERIOLOGY  FOR  NURSES 

ant,  and  a  little  hardier  still  are  the  typhoid,  diphtheria,  and 
tubercle  bacilli  and  the  staphylococci. 

The  "  carrier  "  is  perhaps  one  of  the  most  potent  factors  in 
the  spread  of  disease  and  one  of  the  most  difficult  to  control  since 
he  can  only  be  detected  by  laboratory  examination,  and  even 
when  detected  cannot  always  be  isolated.  Fortunately  in  many 
cases  quarantine  is  not  necessary.  Specific  treatment  and  clean- 
liness may  speedily  cure  the  condition  or  render  him  less  dan- 
gerous. 

Koch's  Postulates.  —  According  to  Koch  an  organism  can  be 
considered  the  causal  agent  of  a  given  disease  only  after  it  has 
fulfilled  certain  requirements:  (1)  it  must  always  be  associated 
with  the  disease;  (2)  be  isolated  in  pure  culture;  (3)  produce 
the  disease  when  inoculated  into  a  healthy  animal,  and  (4)  be 
obtained  again  in  pure  culture.  For  a  long  time  these  conditions 
were  accepted  as  the  only  proof  of  such  a  causal  relationship. 
Recent  studies  in  immunology  and  the  demonstration  of  specific 
serum  reactions  has,  however,  rendered  such  a  procedure  for  the 
most  part  unnecessary.' 


CHAPTER  X 

BACTERIOLOGICAL   EXAMINATIONS 

THE  general  principles  to  be  observed  in  obtaining  material  for 
bacterial  examination  are  (1)  extreme  care  that  the  material  is 
not  contaminated  with  organisms  from  other  sources ;  (2)  that 
the  bacteria  in  the  specimen  are  not  injured  by  a  disinfectant, 
heat,  etc. ;  (3)  that  the  material  is  examined  with  the  least  possible 
delay. 

Whenever  possible  it  is  advisable  to  make  films  or  inoculate 
media  directly  from  the  patient's  body.  If  this  cannot  be  done 
the  material  should  be  placed  in  a  sterile  container  and  sent  as 
quickly  as  possible  to  the  laboratory.  On  no  account  should  a 
disinfectant  be  added.  If  a  short  delay  is  unavoidable  the  speci- 
men should  be  kept  in  the  refrigerator  in  order  that  development 
of  the  organisms  may  be  arrested  and  the  stronger  species  may 
not  thrive  at  the  expense  of  the  weaker.  The  immediate  exami- 
nation of  material  containing  a  great  number  and  variety  of  organ- 
isms cannot  be  over-emphasized.  If,  for  example,  a  specimen  of 
feces  is  to  be  examined  for  typhoid  bacilli  a  delay  of  twenty-four 
hours  may  result  in  their  entire  disappearance  if  they  were  present 
in  small  numbers. 

Material  from  abscesses,  open  wounds,  and  mucous  membranes 
can  best  be  obtained  by  means  of  a  sterile  swab ;  fluid  exudates 
may  be  taken  with  a  sterile  syringe  or  capillary  pipette.  To  make 
the  latter,  a  piece  of  glass  tubing  about  ten  inches  long  is  heated 
in  the  center  in  a  Bunsen  flame  until  it  begins  to  soften,  then  with- 
drawn and  quickly  stretched  until  the  center  has  the  required 
diameter.  The  tube  is  cut  with  a  file  so  as  to  obtain  two  tubes  the 
needed  length.  A  little  plug  of  cotton  is  placed  within  the  large 
ends  and  the  pipettes  are  sterilized  in  a  plugged  test  tube. 

105 


106  BACTERIOLOGY  FOR  NURSES 

Examination  of  Pus.  —  Films  should  first  be  made  and  stained 
with  methylene  blue,  with  carbol  fuchsin,  and  by  Gram's  method. 
Whenever  the  latter  method  is  employed  it  is  advisable  to  smear 
on  the  end  of  the  same  slide  a  small  amount  of  a  known  Gram 
positive  culture  (staphylococcus)  and  of  a  known  Gram  negative 
one  (B.  coli)  as  a  control.  Occasionally  this  preliminary  exami- 
nation reveals  all  it  is  necessary  to  know ;  if  not,  a  loopful  of  pus 
is  inoculated  into  appropriate  media  and  streaks  are  made  on 
agar  and  blood  or  serum  agar  plates.  In  most  cases  it  is  well  to 
make  anaerobic  cultures  also.  When  colonies  have  developed 
on  the  plates,  media  is  inoculated  and  a  film  made  from  each 
variety  present ;  further  methods  of  identification  of  species  have 
already  been  described. 

When  the  only  information  required  is  the  knowledge  of  the 
presence  or  absence  of  a  definite  species,  then  only  the  special 
methods  for  the  detection  of  those  organisms  need  be  employed. 

Nose  and  Throat  Cultures.  —  The  exudate  is  obtained  by  means 
of  a  sterile  swab,  which  is  immediately  replaced  within  its  sterile 
container  if  the  examination  cannot  be  made  at  once.  If  the  ex- 
amination is  to  be  made  for  the  diphtheria  bacillus  the  swab  is 
lightly  smeared  over  a  sterile  slide  and  then  over  the  surface  of 
Loeffler's  serum  media.  The  smear  when  stained  with  Loeffler's 
methylene  blue  may  reveal  the  presence  of  B.  diphtheria?;  if  not, 
the  serum  culture  should  be  incubated  from  twelve  to  eighteen 
hours.  A  film  prepared  from  the  resulting  growth  will  reveal 
the  organisms  if  they  are  present. 

For  septic  sore  throat  the  procedure  is  the  same  save,  in  addition, 
plates  of  blood-smeared  agar  should  be  streaked.  Long  chains  of 
streptococci  will,  however,  generally  be  seen  in  the  original  film. 

For  the  detection  of  tubercle  bacilli  the  procedure  is  the  same 
as  for  sputum. 

In  the  case  of  Vincent's  angina  the  typical  fusiform  bacilli  and 
spirochetes  will  be  seen  in  the  smear.  A  strong  stain  such  as 
gentian  violet  gives  the  best  picture. 

Sputum.  —  Patients  should  be  instructed  to  rinse  the  mouth 
well,  so  that  particles  of  food  may  not  be  mixed  with  the  sputum. 


BACTERIOLOGICAL  EXAMINATIONS  107 

Since  the  early  morning  sputum  coughed  up  from  the  lungs  is 
most  likely  to  contain  the  tubercle  bacilli,  that  should  be  collected 
if  possible  in  a  wide-mouthed  receptacle.  In  making  the  examina- 
tion one  of  the  yellowish  white  cheesy  masses  is  taken  on  a  sterile 
platinum  loop  and  smeared  on  a  glass  slide.  The  film  is  fixed 
by  heat  in  the  usual  manner  and  stained  with  Ziehl-Neelsen's 
carbol  fuchsin. 

If  the  bacilli  in  the  sputum  are  not  sufficiently  numerous  to  be 
detected  by  the  above  method  they  may  sometimes  be  found  if 
a  concentration  be  effected.  Antiformin,  a  patented  preparation 
consisting  of  sodium  hydroxide  and  sodium  hypochlorite,  is  mixed 
with  the  sputum  in  the  proportion  of  about  one  sixth  antiformin 
to  five  sixths  sputum ;  the  mixture  is  thinned  with  a  little  sterile 
water  and  centrifuged.  The  clear  upper  fluid  is  removed,  more 
water  added  to  the  sediment,  and  recentrifuged.  The  upper  fluid 
is  again  removed,  and  the  sediment  smeared  on  to  slides  and 
stained.  The  antiformin  dissolves  the  sputum  and  kills  most  of 
the  bacteria  except  the  tubercle  bacilli. 

If  negative  results  are  obtained  by  both  of  the  above  methods 
the  search  for  the  tubercle  bacilli  may  be  continued  by  means  of 
animal  inoculation.  About  2  c.c.  of  sputum  thinned  with  a  little 
salt  solution  is  injected  subcutaneously,  preferably  in  the  thigh, 
into  a  guinea  pig.  At  the  end  of  four  to  six  weeks  the  animal  will 
probably  die ;  if  not  it  is  best  to  kill  it  by  allowing  it  to  inhale  ether 
or  chloroform.  The  animal  is  autopsied  and  the  peritoneal  nodes, 
the  spleen,  and  the  inguinal  nodes  at  the  site  of  inoculation  removed. 
The  tissue  is  cut  in  small  pieces  with  a  sterile  knife  and  forceps. 
Films  are  made  and  portions  of  the  tissue  are  gently  rubbed  over  the 
surface  of  special  media.  In  this  way  pure  cultures  may  be  ob- 
tained from  material  containing  very  few  tubercle  bacilli  although 
other  organisms  may  be  abundant.  Growth  on  artificial  media 
is  comparatively  slow ;  it  may  not  appear  on  the  inoculated  tubes 
before  the  end  of  seven  or  eight  days. 

Urine.  —  Specimens  of  urine  for  bacteriological  examination 
should  be  taken  by  means  of  a  sterile  catheter  and  passed  into  a 
sterile  container.  The  urine  is  centrifuged,  the  upper  fluid  poured 


108  BACTERIOLOGY   FOR  NURSES 

off,  more  urine  added  to  the  sediment,  and  recentrifuged.  All  the 
fluid  is  then  removed  and  films  and  streak  plates  made  from  the 
sediment.  If  necessary  an  animal  is  inoculated. 

Feces.  —  A  great  variety  of  bacteria  are  present  in  feces.  Very 
few,  however,  are  considered  of  pathological  significance.  The 
different  forms  may  be  isolated  by  inoculating  a  tube  of  broth  with 
a  small  amount  of  feces  and  streaking  pour  plates  with  the  result- 
ing emulsion. 

Bacterial  examination  of  feces  is  generally  made  for  the  isolation 
of  the  typhoid  bacillus,  less  often  for  the  cholera  spirillum.  The 
methods  employed  will  be  considered  in  the  special  sections. 

Examinations  are  occasionally  made  for  the  detection  of  tubercle 
bacilli  in  feces.  It  is  considered  of  doubtful  significance,  however, 
since  they  may  be  present  in  the  feces  of  persons  suffering  from 
pulmonary  tuberculosis  as  a  result  of  swallowing  sputum. 

Ear  Cultures.  —  From  the  discharge  a  culture  is  made  on  Loef- 
fler's  serum  and  also  two  film  preparations;  one  of  the  latter  is 
stained  by  Gram's  method,  the  other  by  a  method  appropriate  for 
the  detection  of  any  special  species  thought  to  be  present.  The 
culture  is  examined  after  eighteen  to  twenty-four  hours'  incuba- 
tion and  if  necessary  the  resulting  growth  is  plated  on  agar  to 
isolate  the  organisms  in  pure  culture  and  prove  their  identity. 

Eye  Cultures.  —  The  procedure  is  the  same  as  above  except 
that  in  addition  to  culturing  on  Loeffler's  serum  cultures  should 
also  be  made  on  blood  agar. 

Blood  Cultures.  —  In  order  to  detect  organisms  occurring  in 
the  blood  in  relatively  small  numbers,  such  as  the  typhoid  bacillus, 
it  is  often  necessary  to  make  blood  cultures.  The  skin  should  be 
thoroughly  cleansed  as  for  a  surgical  operation  and  10  to  15  c.c. 
of  blood  withdrawn  from  the  median  basilic  vein  of  the  arm  by 
means  of  a  sterile  hypodermic  needle  and  syringe.  Whenever 
possible  it  is  best  to  inoculate  the  culture  media  at  the  bedside 
while  the  blood  is  liquid.  If  this  is  not  convenient  coagulation  may 
be  prevented  by  drawing  into  the  syringe  before  the  needle  is  in- 
serted into  the  vein  an  equal  quantity  of  2  per  cent  sodium  citrate 
or  by  immediately  transferring  the  blood  to  a  test  tube  containing 


BACTERIOLOGICAL  EXAMINATIONS  109 

the  sodium  citrate.  Five  c.c.  of  blood  is  added  to  a  flask  con- 
taining 100  c.c.  of  glucose  meat  infusion  broth,  1  c.c.  to  each  of 
two  or  three  test  tubes  of  broth,  and  1  c.c.  to  two  or  three  tubes 
of  glucose  agar,  cooled  to  40°  C.  The  latter  are  thoroughly  mixed 
by  a  rotary  motion  to  avoid  the  formation  of  air  bubbles,  after 
which  the  necks  of  the  tubes  are  flamed  and  the  contents  carefully 
poured  into  Petri  dishes.  This  procedure  not  only  makes  possible 
the  isolation  of  the  organisms,  it  also  enables  a  rough  estimate  to 
be  made  of  the  number  present.  From  the  broth  tubes  other 
tests  may  be  made  if  the  organism  is  obtained  in  pure  culture. 

Solid  Organs.  —  When  solid  organs  are  to  be  examined  about 
one  inch  of  the  surface  is  seared  with  a  hot  spatula  or  scalpel  in 
order  to  kill  all  extraneous  organisms.  An  incision  is  made  in 
the  seared  area  with  a  sterile  scalpel  and  small  quantities  of  the 
fluid  within  the  organ  removed  with  a  platinum  loop  and  trans- 
ferred to  suitable  media. 

Examination  of  Bacteria  in  Tissues.  —  In  order  to  examine 
bacteria  in  body  tissues  the  latter  must  be  fixed,  hardened,  and 
cut  in  extremely  thin  section. 

Fixation  consists  in  treating  the  tissue  in  such  a  manner  that  it 
will  preserve  as  far  as  possible  its  condition  at  the  time  of  removal 
from  the  body ;  hardening  renders  it  firm  enough  to  be  cut  in  very 
thin  sections  with  a  microtome. 

The  best  results  are  obtained  by  embedding  in  solid  paraffin. 
Impregnation  with  paraffin  in  the  melted  state  gives,  when  solidi- 
fied, support  to  the  tissue  elements  and  greatly  facilitates  the 
cutting  process.  After  hardening,  the  tissue  is  dehydrated  and 
then  completely  permeated  by  a  solvent  of  paraffin  which  will 
rid  the  tissue  of  the  dehydrating  fluid  and  at  the  same  time  make 
possible  the  entrance  of  the  paraffin.  The  solvents  in  general  use 
are  chloroform,  cedar  oil,  xylol,  and  turpentine. 

Several  methods  of  more  or  less  value  have  been  devised.  The 
following  is  simple  and  gives  good  results. 

Fixation.  —  As  soon  as  possible  after  removal  from  the  body 
small  pieces  of  tissue  about  -j-  inch  by  i  inch  are  cut  and  placed 
in  the  fixative  prepared  as  follows:  To  a  heated  0.75  per  cent 


110  BACTERIOLOGY  FOR  NURSES 

solution  of  sodium  chloride  add  sufficient  bichloride  of  mercury 
to  make  a  saturated  solution.  Small  pieces  of  tissue  are  immersed 
in  the  solution  for  about  ten  hours ;  for  larger  pieces  twenty-four 
hours  is  necessary.  They  are  then  tied  in  a  piece  of  gauze  and 
placed  in  a  stream  of  running  water  for  from  twelve  to  twenty 
hours  according  to  their  size  in  order  to  wash  out  the  excess  of 
bichloride  of  mercury. 

Hardening.  —  The  pieces  of  tissue  are  placed  for  twenty-four 
hours  successively  in  each  of  the  following  strengths  of  ethyl 
alcohol ;  30  per  cent,  60  per  cent,  90  per  cent,  and  finally  absolute 
alcohol.  They  are  then  ready  to  be  embedded  in  paraffin.  If 
only  a  minute  particle  of  tissue  is  to  be  examined  for  diagnosis 
all  the  stages  may  be  compressed  into  twenty-four  hours. 

Embedding  in  Paraffin.  —  The  tissue  is  transferred  to  (1)  cedar 
oil  or  xylol  until  translucent;  (2)  equal  parts  of  cedar  oil  and 
paraffin  at  37°  C.  for  two  hours ;  (3)  melted  paraffin  at  52°  C. 
for  four  hours ;  (4)  each  piece  of  tissue  is  removed  from  the  hot 
paraffin  by  means  of  forceps  and  placed  in  very  small  tin  or  paper 
box,  after  which  it  is  surrounded  with  the  melted  paraffin.  The 
paraffin  used  should  be  one  that  has  a  melting  point  about  52°  C. 
When  the  block  is  cold  the  edges  are  pared  and  the  preparation  is 
ready  for  sectioning. 

Cutting  of  Paraffin  Sections.  —  A  microtome  is  generally  used 
for  this  purpose.  Each  section  should  be  as  thin  as  possible. 
When  cut  the  sections  are  floated  on  the  surface  of  a  glass  of  warm 
water  kept  at  about  40°  C.,  where  in  a  short  time  they  become 
perfectly  flat. 

Fixation  on  Slides.  —  A  clean  slide  is  thrust  obliquely  into 
the  water  below  the  section,  a  corner  of  the  section  is  held  on  to 
it  with  a  needle,  and  the  slide  is  withdrawn.  The  surplus  water 
is  wiped  off  with  a  cloth,  the  section  carefully  adjusted  by  means 
of  a  camel's  hair  brush,  and  the  slide  placed  on  a  support  with 
the  section  side  downward  in  an  incubator  for  from  twelve  to  fifteen 
hours ;  it  will  then  be  sufficiently  fixed  for  staining.  Before  stain- 
ing, however,  the  paraffin  must  be  removed  by  dropping  on  to  it 
a  little  xylol.  When  the  paraffin  is  dissolved  the  xylol  is  removed 


BACTERIOLOGICAL  EXAMINATIONS  111 

by  gently  wiping  with  filter  paper,  after  which  a  little  absolute 
alcohol  is  dropped  on  to  the  section.  If  any  crystals  of  bichloride 
of  mercury  are  noticed  they  may  be  removed  before  staining  by 
immersing  the  slides  for  a  few  minutes  in  equal  parts  of  Gram's 
iodine  solution  and  water  and  then  washing  out  the  iodine  with 
alcohol. 


CHAPTER  XI 

BACTERIAL  TOXINS  AND  ANTITOXINS 

THE  ability  of  bacteria  to  produce  toxin  is  their  chief  weapon 
of  offense  in  the  production  of  disease.  Poisonous  products  such 
as  acids,  alkalies,  hydrogen  sulphid,  etc.,  result  from  the  growth 
of  many  species,  and  such  substances  may  play  a  minor  role  in 
diseased  conditions.  They  are,  however,  totally  different  to  toxins. 
Fortunately  only  a  comparatively  few  of  the  vast  family  of  micro- 
organisms are  able  to  produce  the  latter. 

Bacterial  toxins  are  divided  into  two  classes:  (1)  those  inti- 
mately connected  with  the  bacterial  protoplasm  and  liberated 
only  after  the  death  of  the  organism,  and  (2)  those  which  result 
from  living  bacterial  activity,  are  soluble,  and  pass  out  from  the 
organism  into  the  surrounding  medium.  The  former  are  spoken 
of  as  endotoxins,  the  latter  as  exotoxins. 

Endotoxins.  —  When  the  dead  bodies  of  certain  bacteria  are  in- 
jected into  animals  toxic  effects  are  frequently  observed.  For 
example,  if  tubercle  bacilli  are  killed  by  heat  and  injected  into  the 
tissues  of  a  susceptible  animal  tubercular  nodules  will  be  found 
to  have  formed  around  the  point  of  injection ;  the  dead  cells  of 
typhoid  bacilli  and  cholera  spirilla  likewise  give  rise  to  pathogenic 
effects.  So  far  it  has  been  impossible  to  obtain  these  toxins  apart 
from  the  bacterial  protoplasm.  Since  the  death  of  bacteria  must 
be  constantly  occurring  both  in  culture  medium  and  in  the  body 
during  infection  it  is  reasonable  to  suppose  that  these  dead  bac- 
terial cells  are  disintegrated  and  the  endotoxin  thus  set  free.  ,  It 
is  believed  that  the  living  members  secrete  a  ferment  that  has  a 
solvent  action  upon  the  dead  organisms  and  that  upon  this  process 
of  autolysis  the  liberation  of  the  poison  depends.  However  this 
may  be,  it  is  definitely  known  that  the  body  cells  secrete  a  substance 
which  has  a  strongly  lytic  action  upon  bacteria. 

112 


BACTERIOLOGICAL  TOXINS  AND  ANTITOXINS     113 

The  action  of  the  endotoxins  on  the  body  tissues  differs  from 
that  of  the  exotoxins  in  that  they  do  not  give  rise  to  symptoms 
of  a  specific  character.  There  is  no  definite  period  before 
symptoms  appear,  nor  do  they  stimulate  the  body  cells  to  the 
production  of  antitoxin^.  Certain  protective  substances  are  elabo- 
rated against  them,  but  they  are  of  a  totally  different  nature  to 
antitoxin. 

Aggressins.  —  In  certain  cases  it  is  difficult  to  understand  a 
result  of  bacterial  growth  which  is  due  neither  to  endotoxins  nor 
exotoxins  but  to  some  other  substance  which  aids  the  organisms 
in  combating  the  body  defenses.  If,  for  instance,  tuberculous 
exudate  is  removed  from  an  animal  suffering  from  the  disease,  and 
after  sterilization  it  is  injected  into  a  healthy  guinea  pig,  it  has 
practically  no  effect.  If  into  a  second  guinea  pig  tubercle  bacilli 
are  injected  the  usual  lesions  appear  in  from  four  to  six  weeks.  If, 
however,  into  a  third  guinea  pig  the  sterile  exudate  and  a  com- 
paratively small  quantity  of  tubercle  bacilli  are  injected  death 
follows  within  twenty-four  hours,  indicating  that  the  exudate 
had  a  paralyzing  effect  upon  the  defensive  forces  of  the  animal 
cells,  thus  greatly  increasing  the  virulence  of  the  bacilli.  That  the 
effect  is  not  produced  by  endotoxin  in  the  sterilized  exudate  is 
proved  by  the  fact  that  the  exudate  alone  produced  no  reaction. 
Similar  results  are  obtained  with  other  organisms  such  as  typhoid 
and  dysentery  bacilli,  cholera  spirilla,  etc.  It  has  been  assumed 
therefore  that  the  exudate  contains  a  substance  which  enables  the 
bacilli  to  become  more  aggressive.  Consequently  this  hypotheti- 
cal substance  has  received  the  name  aggressin.  It  is  believed  that 
it  has  a  paralyzing  effect  upon  the  polynuclear  leukocytes,  which, 
as  we  shall  see  later,  constitute  one  of  the  body's  strongest  defenses. 
In  general  the  production  of  aggressins  is  most  abundant  when  the 
body  resistance  is  greatest.  It  is  thought  by  certain  authorities 
that  aggressins  are  secreted  by  bacteria  as  a  means  of  protection 
against  the  opposing  forces  of  the  animal  cells,  just  as  the  body 
cells  produce  antitoxin  to  neutralize  bacterial  toxin.  According  to 
this  theory  toxins  may  be  considered  as  the  offensive  agents  of 
bacteria  and  aggressins  as  their  defensive  weapons. 


114  BACTERIOLOGY  FOR  NURSES 

Exotoxins.  —  There  is  evidence  that  the  exotoxins  or  true  toxins 
are  of  a  protein  nature,  although  nothing  is  known  of  their  chemi- 
cal structure;  nor  is  it  known  whether  they  are  secretory  or 
excretory  products.  They  are,  however,  undoubtedly  produced 
by  bacteria  during  their  growth.  They  are  poisonous  in  minute 
doses ;  reproduce  characteristic  symptoms  and  lesions  of  the  disease 
after  a  period  of  incubation ;  are  soluble  in  water ;  are  destroyed 
by  heat,  and  in  the  animal  body  stimulate  the  cells  to  produce 
defensive  substances  which  neutralize  them,  —  so-called  antitoxins. 

The  three  well-known  exogenous  toxins  are  those  produced 
by  B.  diphtherise,  B.  tetani,  and  B.  botulismus.  They  can  be 
produced  apart  from  the  body  by  growing  the  organisms  on  cul- 
ture medium;  the  toxin  passes  from  the  bacterial  cell  and  is 
diffused  throughout  the  substance  on  which  the  bacteria  are  grow- 
ing. If  broth  is  employed  the  toxin  can  be  obtained  germ  free 
by  passing  the  broth  through  a  porcelain  filter.  All  attempts 
to  separate  the  toxin  from  the  broth  have  failed.  The  only  way 
its  presence  can  be  recognized  is  through  its  effects  on  animals. 
By  this  means  it  is  possible  not  only  to  determine  the  presence  of 
the  poison  but  also  its  strength. 

As  already  stated,  true  toxins  are  poisonous  in  exceedingly 
small  amounts.  Tetanus  toxins  have  been  prepared  from  cultures 
of  the  bacilli  so  strong  that  .0002  gram  would  be  a  fatal  dose  for 
a  man  weighing  140  pounds. 

Another  of  the  most  distinguishing  features  of  the  true  toxins 
is  their  ability  to  produce  all  the  characteristic  symptoms  that 
arise  when  the  disease  is  naturally  contracted  and  the  specific 
organisms  are  present.  Thus  after  an  injection  of  diphtheria  toxin 
an  animal  will  show  depression,  necrosis  of  the  tissue  at  the  site  of 
inoculation,  post-diphtheria  paralysis,  etc. 

Small  amounts  of  diphtheria  or  tetanus  toxin  prove  fatal  when 
circulating  in  the  blood  stream ;  larger  quantities,  however,  when 
taken  by  mouth  are  not  injurious  because  they  are  immediately 
destroyed  by  the  digestive  juices.  On  the  other  hand,  the  toxin 
of  botulism  is  absorbed  by  the  mucous  membranes  of  the  intes- 
tines and  by  this  route  gains  entrance  to  the  circulation.  A  strange 


BACTERIOLOGICAL  TOXINS  AND  ANTITOXINS     115 

fact  in  connection  with  the  latter  toxin  is  that  it  is  produced  out- 
side of  the  body  and  not  within  it.  B.  botulismus  will  grow  and 
produce  its  poison  on  almost  any  form  of  protein.  Sausages  are 
probably  the  most  frequent  source  of  botulism.  When  the  food 
enters  the  intestines,  the  poison  which  is  intimately  mixed  with 
it  is  absorbed  and  the  characteristic  disease  is  produced.  As  in 
the  case  of  diphtheria  and  tetanus  a  specific  antitoxin  is  formed 
by  the  body  cells  seeking  to  protect  themselves  from  this  par- 
ticular poison. 

The  difference  between  botulism  and  ptomain  poisoning  should 
be  clearly  understood ;  botulism  results  from  eating  food  contain- 
ing a  true  toxin  elaborated  by  the  Bacillus  botulismus.  Ptomain 
poisoning,  on  the  other  hand,  results  from  the  ingestion  of  food 
that  has  been  decomposed  perhaps  by  several  species  of  bacteria 
into  simple  but  poisonous  compounds.  In  the  former  case  the 
poison  is  produced  by  bacteria;  in  the  latter  the  poison  is  the 
disintegrated  meat,  fish,  cheese,  or  whatever  the  protein  may 
be.  Another  important  difference  between  the  true  toxins  and 
ptomains  is  that  the  latter  do  not  stimulate  the  body  cells  to 
the  production  of  antitoxin. 

Phytotoxins.  —  Substances  resembling  in  action  the  bacterial 
toxins  occur  in  the  seeds  of  some  of  the  higher  plants;  among 
them  are  ricin  from  the  castor  oil  bean  and  abrin  from  the  jequirty 
bean.  These  toxins  of  vegetable  origin  are,  like  the  bacterial 
toxins,  exceedingly  poisonous  in  small  amounts,  act  only  after  a 
period  of  incubation,  are  destroyed  by  heat,  and  produce  specific 
antitoxin. 

Zootoxins.  —  Closely  corresponding  poisons  are  found  in  the 
blood  and  secretions  of  a  number  of  animals.  Snake  venom,  the 
poisons  of  scorpions  and  spiders,  as  well  as  poisonous  substances 
present  in  eel  blood,  are  well-known  examples.  They,  too,  like 
the  true  bacterial  toxins  cause  the  production  of  antitoxins  when 
injected  into  the  body  of  another  animal. 

Diphtheria  Toxin.  —  Diphtheria  bacilli  usually  find  lodgment 
upon  some  portion  of  the  upper  respiratory  tract,  either  the  tonsils, 
the  larynx,  or  not  infrequently  on  the  nasal  mucous  membrane. 


116  BACTERIOLOGY  FOR  NURSES 

Occasionally  they  find  conditions  suitable  for  growth  on  an  abrasion 
of  the  skin,  in  the  vulva,  or  on  the  conjunctiva.  If  they  are  able 
to  develop  they  secrete  during  their  growth  the  toxin  which  de- 
stroys the  epithelial  cells,  and  from  this  area  of  infection  the  poison 
is  absorbed  and  carried  to  all  parts  of  the  body.  Other  micro- 
organisms such  as  pneumococci  and  streptococci,  which  may  be 
harmless  on  intact  healthy  membranes,  readily  grow  in  the  necrotic 
tissue  and  may  add  to  the  severity  of  the  local  lesion  and  the  gen- 
eral toxic  condition. 

Diphtheria  bacilli  rarely  enter  the  blood  stream ;  they  usually 
remain  localized,  and  only  their  toxin  affects  the  susceptible  body 
tissues.  The  outcome  of  the  infection  depends  largely  upon  the 
amount  and  the  strength  of  the  toxin  absorbed  and  the  quantity 
of  antitoxin  present  in  the  blood.  The  amount  of  tissue  involved 
in  the  nose  or  throat  is  not  always  an  indication  of  the  severity  of 
the  disease ;  virulent  bacilli  in  a  small  patch  may  produce  more 
toxin  than  less  virulent  ones  occupying  a  larger  area.  As  a  rule, 
the  patient  who  has  most  antitoxin,  whether  naturally  present 
or  gained  as  a  result  of  a  previous  attack  of  the  disease  or  an  in- 
jection of  antitoxin,  will  be  least  affected  even  though  the  bacilli 
be  extremely  virulent. 

Twenty  years  ago  diphtheria  was  one  of  the  most  dreaded 
diseases ;  it  had  a  mortality  of  at  least  30  per  cent.  About  1888 
Roux  and  Yersin  discovered  that  it  was  produced  by  a  soluble 
poison ;  two  years  later  von  Behring  demonstrated  that  circulating 
in  the  blood  of  all  animals  recovering  from  an  attack  of  the  disease 
there  was  a  certain  substance,  antitoxin,  which  rendered  the  poison 
harmless.  It  seemed  evident  that  this  substance  must  be  elabo- 
rated by  the  body  cells  to  save  themselves  from  being  attacked 
by  the  toxin.  The  idea  was  then  conceived  that  if  a  serum  rich 
in  antitoxin  could  be  injected  into  a  person  suffering  from  diph- 
theria it  would  afford  protection  by  neutralizing  the  toxin  until 
the  cells  of  the  patient  could  respond  sufficiently  to  produce  their 
own  antitoxin  in  large  enough  amounts.  At  first  the  results  were 
not  as  satisfactory  as  expected  because  the  serum  was  not  powerful 
enough.  By  1896,  however,  it  had  been  sufficiently  perfected  to 


BACTERIOLOGICAL  TOXINS  AND  ANTITOXINS     117 

cause  a  marked  decrease  in  the  mortality  from  diphtheria  in  those 
communities  in  which  it  had  been  used.  Since  then  thousands 
of  lives  have  been  saved  by  its  means.  Records  show  that  when 
antitoxin  is  used  on  the  first  day  of  the  disease  practically  no  mor- 
tality occurs. 

Production  of  Antitoxin  for  Therapeutic  Purposes.  —  Toxin 
is  first  prepared  by  cultivating  a  virulent  strain  of  the  bacilli  in 
a  suitable  broth  medium  for  ten  days  or  two  weeks.  The  culture 
is  then  passed  through  a  porcelain  filter  which  retains  the  bacilli 
and  allows  the  strong  toxin  solution  to  pass  through.  The  potency 
of  this  toxin  is  estimated  by  injecting  carefully  measured  doses 
into  a  series  of  guinea  pigs.  Usually  about  -^ro  c.c.  of  a  moderately 
strong  toxin  is  fatal.  The  smallest  amount  of  toxin  that  will 
kill  in  four  days  a  guinea  pig  weighing  250  grams  is  spoken  of  as 
the  toxin  unit  or  minimum  lethal  dose.  This  preliminary  titration 
serves  to  indicate  how  much  toxin  can  with  safety  be  injected  into 
a  horse  of  much  heavier  body  weight. 

Horses  are  chosen  for  the  production  of  commercial  antitoxin 
partly  because  large  quantities  of  serum  can  be  obtained  from  each 
animal  and  partly  because  the  serum  is  normally  of  a  bland  nature. 
Strong  healthy  horses  which  have  been  proved  to  be  free  from 
tuberculosis  and  glanders  are  injected  with  gradually  increasing 
doses  of  the  toxin ;  the  first  doses  are  usually  guarded  by  an  injec- 
tion of  antitoxin  given  at  the  same  time.  As  soon  as  the  reaction 
passes  and  the  temperature  becomes  normal  a  slightly  larger  dose  of 
toxin  is  given,  until  by  the  end  of  about  eight  weeks  the  horse  can 
tolerate  many  times  the  amount  received  on  the  first  day.  Injec- 
tions are  continued  and  weekly  tests  are  made  of  the  serum  until 
it  is  found  that  the  amount  of  antitoxin  no  longer  increases.  At  the 
end  of  from  four  to  six  months  the  serum  may  contain  from  700 
to  1000  units  of  antitoxin  per  cubic  centimeter.  When  no  further 
increase  appears  the  horse  is  bled  aseptically  from  the  jugular 
vein  and  the  blood  stored  in  the  refrigerator  for  two  days  in  order 
that  the  serum  may  separate  from  the  clot.  At  the  end  of  this 
period  the  serum  is  siphoned  off,  and  after  the  addition  of  a  small 
amount  of  an  antiseptic  as  a  preservative  it  is  ready  for  use.  A 


118  BACTERIOLOGY  FOR  NURSES 

method  of  concentrating  such  a  serum  has  been  devised  which 
not  only  reduces  its  bulk  but  is  said  to  lessen  the  probability  of 
serum  sickness. 

After  a  short  period  of  rest  a  horse  that  has  been  used  for  the 
production  of  antitoxin  may  be  used  in  the  same  manner  for  a 
further  supply.  Given  three  months  rest  each  year  a  horse  is 
said  to  furnish  a  high-grade  serum  for  three  or  four  years. 

Standardization  of  Antitoxin.  —  At  first  antitoxin  serum  was 
administered  in  so  many  cubic  centimeter  doses,  but  since  some 
horses  produce  a  much  more  powerful  serum  than  others  the  results 
were  altogether  irregular.  The  adoption  of  a  standard  unit, 
therefore,  was  deemed  necessary  in  order  to  obtain  a  certain 
degree  of  accuracy  in  dosage.  As  a  result  the  standard  unit  of 
antitoxin  was  fixed  as  the  smallest  amount  that  will  just  neutralize 
one  hundred  times  the  amount  of  toxin  that  will  kill  a  guinea  pig 
weighing  250  grams  in  four  days. 

In  order  to  standardize  an  antitoxin  it  is  necessary  to  have  a 
standard  toxin  against  which  to  test  it.  Since  toxin  quickly  dete- 
riorates a  standard  antitoxin  is  supplied  in  small  quantities  by  the 
Hygienic  Laboratory  at  Washington  to  the  various  manufacturers 
for  this  purpose.  A  series  of  guinea  pigs  each  weighing  about  250 
grams  are  inoculated  with  1  BT&»  of  the  standard  antitoxin  plus 
varying  amounts  of  toxin.  In  this  way  the  L+  (limes  death) 
dose,  which  is  the  amount  of  toxin  plus.  1  dc.  of  antitoxin  required 
to  kill  a  guinea  pig  in  four  days,  is  obtained.  Having  determined 
the  dose  of  toxin  that  just  neutralizes  the  standard  antitoxin  this 
constant  dose  of  toxin  plus  increasing  amounts  of  the  new  antitoxin 
is  injected  into  another  series  of  250  gram  guinea  pigs.  At  the 
end  of  four  days  it  will  be  found  that  those  receiving  the  smallest 
amounts  of  antitoxin  have  died  and  that  the  larger  amounts  have 
protected  the  animals.  The  strength  of  the  antitoxin  is  estimated 
from  the  smallest  protective  amount.  Thus  if  a  guinea  pig  receiving 
.003  c.c.  of  serum  had  died  and  the  next  of  the  series  receiving 
.004  c.c.  had  died  also,  but  the  third  which  had  received  .005  c.c. 
and  likewise  all  the  others  receiving  larger  doses  had  been  protected, 
then  the  serum  would  be  said  to  contain  200  units  of  antitoxin 


per  c.c.  One  two-hundredth  of  a  c.c.  or  .005  c.c.  neutralized  the 
standardized  toxin ;  therefore  each  cubic  centimeter  of  the  serum 
contained  200  units  of  antitoxin.  '" 

Antitoxins  are  fairly  stable  bodies.  When  kept  in  a  cool,  dark 
place  they  may  be  preserved  for  a  year  or  more  with  very  little 
deterioration.  It  is  customary  for  manufacturers  to  place  a  label 
on  each  package  bearing  a  date  beyond  which  the  serum  is  not 
guaranteed  to  contain  the  original  amount  of  antitoxin. 

The  most  important  point  in  the  administration  of  antitoxin 
is  that  it  be  given  as  soon  as  possible  after  infection  has  occurred ; 
2000  units  on  the  first  day  that  symptoms  appear  is  considered 
more  efficacious  than  5000  units  on  the  second  day.  A  total  of 
10,000  or  even  20,000  units  in  severe  cases  is  sometimes  given. 

Prophylactic  Immunization  against  Diphtheria.  —  The  sub- 
cutaneous injection  of  antitoxin  will  afford  protection  for  a  limited 
period  to  susceptible  persons  exposed  to  infection.  The  doses  are 
relatively  small  and  the  injection  does  not  usually  produce  any 
ill  effects  save  a  little  soreness  at  the  site  of  inoculation.  Un- 
fortunately antitoxin  injected  into  the  body  is  eliminated  rather 
rapidly,  so  that  protection  thus  gained  lasts  only  from  two  to  four 
weeks.  As  a  prophylactic  measure  children  under  one  year  are  usu- 
ally given  500  units,  older  children  and  adults  1000  to  1500  units. 

Seeing  that  antitoxin  produced  by  the  body  cells  in  direct 
response  to  the  presence  of  toxin  remains  for  a  much  Ic-nger  period 
in  the  body  than  that  injected  from  another  animal,  a  method  has 
been  advocated  whereby  the  body  cells  may  be  stimulated  to  pro- 
duce their  own  antitoxin  in  sufficient  amounts  to  protect  them- 
selves in  case  of  exposure  to  diphtheria.  A  mixture  of  toxin  and 
antitoxin  known  as  T-A  is  injected  in  graded  doses  subcutaneously. 
The  method  is  based  upon  the  principle  that  the  union  of  toxin 
and  antitoxin  is  not  stable,  and  when  a  neutral  mixture  of  the  two 
is  injected  sufficient  toxin  may  be  dissociated  to  stimulate  the  body 
cells  to  produce  their  own  antitoxin. 

Schick  Test.  —  In  order  to  determine  whether  a  prophylactic 
dose  of  antitoxin  is  necessary  in  case  of  exposure  to  diphtheria 
Schick  has  devised  a  simple  skin  test  for  detecting  the  presence 


120  BACTERIOLOGY  FOR  NURSES 

of  natural  antitoxin  in  the  blood.  A  minute  amount  of  toxin 
(about  one  fifth  of  the  minimum  lethal  dose  for  a  guinea  pig) 
is  injected  intradermically.  If  the  person  receiving  the  toxin 
possess  an  amount  of  antitoxin  equal  to  at  least  one  thirtieth  of  a 
unit  in  each  cubic  centimeter  of  blood  the  injected  toxin  is  neu- 
tralized and  no  reaction  appears ;  if,  on  the  other  hand,  he  has  no 
antitoxin,  the  toxin  acts  as  an  irritant  to  the  skin  and  in  from 
twenty  to  forty-eight  hours  produces  a  small  inflamed  area.  This 
positive  reaction  indicates  that  the  person  has  no  natural  antitoxin 
and  therefore  that  he  is  susceptible  to  the  disease ;  conversely,  a 
negative  reaction  indicates  that  an  individual  has  in  all  probability 
sufficient  natural  antitoxin  to  protect  him,  even  in  case  of  exposure, 
and  a  prophylactic  dose  is  unnecessary. 

Tetanus  Toxin.  —  Tetanus,  like  diphtheria,  is  a  local  infection 
characterized  by  a  general  toxemia.  The  bacilli  and  spores  never 
gain  access  to  the  blood ;  they  remain  at  site  of  infection  where 
they  produce  their  toxin,  which  when  absorbed  is  responsible  for 
the  disease.  The  blood,  then,  usually  contains  tetanus  toxin,  but 
is  sterile  so  far  as  the  organisms  are  concerned.  Two  different 
poisonous  substances  have  been  shown  to  exist  in  tetanus  toxin : 

(1)  tetanospasmin,  which  has  a  special  affinity  for  nerve  tissue  and 
to  the  action  of  which  the  characteristic  symptoms  are  due,  and 

(2)  tetanolysin,  a  substance  of  probably  much  less  importance 
which  attacks  the  red  blood  corpuscles,  ruptures  the  envelope,  and 
liberates  the  contents,  giving  rise  to  a  more  or  less  anemic  condition. 

One  of  the  greatest  dangers  of  tetanus  lies  in  the  fact  that 
while  the  local  lesion  may  show  no  sign  of  disturbance  the  central 
nervous  system  may  suddenly  develop  symptoms  of  poisoning. 
The  toxin  is  produced  during  or  soon  after  the  first  twenty-four 
hours  and  from  the  point  of  infection  it  passes  rapidly  into  the 
blood  and  lymph  stream  and  according  to  certain  authorities 
is  absorbed  by  the  end  plates  of  the  motor  nerves  and  quickly 
travels  along  the  axis  cylinders  to  the  spinal  cord.  The  more  gen- 
eral opinion,  however,  is  that  the  toxin  passes  by  way  of  the  lym- 
phatics of  the  nerves  and  not  by  way  of  the  axis  cylinders.  So 
great  is  the  affinity  of  tetanus  toxin  for  nerve  tissue  that  once 


BACTERIOLOGICAL  TOXINS  AND   ANTITOXINS     121 

united  with  the  nerve  cells  it  is  difficult  or  impossible  to  effect  its 
neutralization ;  hence  the  greatest  value  of  tetanus  antitoxin  lies 
in  its  administration  as  a  prophylactic.  As  a  prophylactic  remedy 
it  is  even  of  more  value  than  diphtheria  antitoxin ;  therapeutically, 
it  is  less  beneficial,  since  the  tetanus  toxin  combines  so  rapidly  and 
firmly  with  the  nerve  cells.  Recently,  however,  a  method  has 
been  employed  which  has  met  with  considerable  success.  A  mod- 
erate amount  of  spinal  fluid  is  removed  by  a  spinal  puncture 
and  from  3000  to  5000  units  of  tetanus  antitoxin  in  a  volume  of 
from  3  to  10  c.c.  of  normal  salt  solution  is  injected  slowly  by 
gravity;  at  the  same  time  10,000  units  are  given  intravenously 
or  intramuscularly.1  As  a  prophylactic  measure  from  1000  to 
1500  units  are  injected  intramuscularly. 

It  is  generally  agreed  that  since  tetanus  is  almost  invariably 
associated  with  a  wound,  proper  treatment  of  the  original  lesion 
combined  with  the  immediate  administration  of  antitoxin  will 
surely  prevent  its  development. 

Production  of  Tetanus  Antitoxin.  —  As  in  the  case  of  diphtheria 
a  strong  toxin  is  first  prepared.  Suitable  broth  is  inoculated  with 
tetanus  bacilli  and  the  organisms  are  grown  anaerobically  for  two 
weeks  at  37°  C.  At  the  end  of  this  period  the  toxin  broth  is 
separated  from  the  bacilli  by  filtration  and  its  strength  is  deter- 
mined by  the  same  method  as  that  employed  for  diphtheria  toxin, 
except  in  this  case  heavier  guinea  pigs  are  used.  Those  weighing 
about  350  grams  are  chosen.  The  immunization  of  horses  and 
procuring  of  serum  are  also  conducted  in  much  the  same  way  as 
for  the  production  of  diphtheria  antitoxin. 

For  the  standardization  of  the  new  antitoxin  a  standard  toxin 
with  which  to  test  it  is  obtained  from  the  Hygiene  Laboratory  at 
Washington.  The  unit  employed  is  not  quite  the  same  as  that 
adopted  for  diphtheria.  A  unit  of  diphtheria  antitoxin  is  defined 
as  the  amount  of  antitoxin  that  will  just  neutralize  100  minimum 
fatal  doses  of  toxin  for  a  250  gram  guinea  pig.  A  unit  of  tetanus 
antitoxin  is  defined  as  the  amount  of  antitoxin  that  will  just  neutralize 
1000  fatal  doses  of  toxin  for  a  350  gram  guinea  pig. 

1  Park  and  Nichol,  Jour,  of  the  A.  M.  A.,  Vol  63,  July,  1917,  p.  325. 


CHAPTER  XII 
IMMUNITY 

FOR  many  years  it  has  been  observed  that  one  attack  of  most 
of  the  infectious  diseases  will  protect  an  individual  against  subse- 
quent attack,  or  at  least  future  attacks  will  be  of  a  less  severe  nature. 
Crude  attempts  were  frequently  made  by  primitive  people  to  ob- 
tain this  protection.  Thus  South  African  tribes  tried  to  defend 
themselves  against  snake  bites  by  using  a  mixture  of  snake  venom 
and  gum ;  in  the  East  in  order  to  obtain  protection  against  a  severe 
attack  of  smallpox  people  deliberately  placed  themselves  in  con- 
tact with  a  mild  case,  hoping  to  contract  the  disease  in  a  similar 
form  and  thus  obtain  protection  against  a  disfiguring  and  probably 
fatal  form. 

The  object  of  these  procedures  was  to  obtain  resistance  or 
immunity.  The  term  immunity  in  its  broadest  sense  may  be  ap- 
plied to  resistance  in  general.  Ordinarily,  however,  it  is  applied 
to  the  power  of  resisting  disease  which  certain  forms  of  life  possess. 
Health  is  a  condition  of  immunity.  So  long  as  we  are  alive  and 
our  body  cells  can  continue  to  manufacture  their  specific  protective 
substances  the  bacteria  on  our  skin  and  in  our  intestinal  tracts 
can  do  no  harm,  but  the  moment  we  die  they  penetrate  our  tissues 
and  disintegrate  them. 

Immunity  is  of  course  the  contrary  condition  to  susceptibility.  It 
is  not  confined  to  the  animal  kingdom  alone ;  it  occurs  also  among 
plants.  The  theory  of  Welch  attributes  its  possession  even  to 
bacteria ;  thus  man  is  susceptible  to  the  typhoid  bacillus  because 
the  typhoid  bacillus  is  immune  to  man ;  conversely  man  is  immune 
to  the  hay  bacillus  because  the  hay  bacillus  is  susceptible  to  man. 

Pasteur  tried  to  explain  the  production  of  immunity  by  his 
"  exhaustion  theory,"  a  theory  now  entirely  disproved.  He  be- 

122 


IMMUNITY  123 

lieved  that  as  bacteria  developed  in  the  tissues  they  used  up 
some  substances  necessary  to  their  existence,  and  that  when  this 
substance  was  exhausted  they  could  no  longer  grow  for  lack  of 
proper  food.  Chauveau  took  an  exactly  opposite  view  and  pro- 
posed his  "  retention  theory."  He  suggested  that  as  in  a  test 
tube  bacterial  growth  may  cease  because  the  organisms  have  pro- 
duced substances  harmful  to  themselves  and  not  because  the  food 
supply  is  exhausted,  so  in  the  body  these  substances  might  be 
formed  and  retained  there  and  prevent  the  future  development 
of  the  organism.  Both  of  these  theories  are  now  only  of  historic 
interest.  It  has  been  proved  that  the  production  of  immunity  is  a 
much  more  complicated  problem,  resembling  rather  a  battle  be- 
tween an  invading  army  and  defensive  forces,  during  which  both 
parties  are  extremely  active. 

When  bacteria  have  succeeded  in  overcoming  the  normal  de- 
fenses of  the  body  and  have  invaded  the  tissues  the  body  cells 
are  by  no  means  overcome ;  the  battle  is  really  only  just  about  to 
begin.  The  presence  of  the  invaders  stimulates  the  body  cells  to 
defend  themselves  by  actively  producing  substances  termed 
antibodies,  by  means  of  which  they  endeavor  to  rid  themselves 
of  the  invading  force  or  neutralize  their  products. 

Antigens  and  Antibodies.  —  Whatever  offensive  weapon  the 
parasite  may  produce,  the  body  cells  manufacture  a  substance 
especially  prepared  to  combat  it.  If  the  toxin  of  a  microorganism 
is  its  chief  means  of  inflicting  injury,  such  for  instance  as  the 
soluble  toxin  of  the  diphtheria  bacillus,  the  body  cells  produce 
an  antitoxin  to  neutralize  it ;  if  it  is  necessary  to  destroy  the  bac- 
teria themselves  then  a  substance,  bacteriolysin,  is  poured  out 
on  the  invaders,  which  dissolves  them.  In  certain  infections,  and 
particularly  those  due  to  pyogenic  cocci,  the  polynuclear  leukocytes 
and  certain  other  cells  engulf  the  organisms  and  carry  them  off 
bodily.  Another  antibody,  opsonin,  appears  to  aid  the  leukocytes 
in  their  work. 

The  term  antigen  is  generally  applied  to  all  substances  that 
cause  the  formation  and  appearance  of  antibodies  in  the  body 
fluids.  This  power  is  not  confined  to  bacteria  and  their  toxins, 


124  BACTERIOLOGY  FOR  NURSES 

but  is  shared,  as  we  have  seen,  by  certain  substances  in  plants 
(phytotoxins)  and  poisonous  secretions  of  certain  animals  (zoo- 
toxins).  Red  blood  corpuscles,  serum  of  different  animals,  egg 
albumen,  milk,  etc.,  when  inoculated  into  the  tissues,  are  treated 
as  foreign  substances  and  are  disposed  of  as  quickly  as  possible 
by  antibodies  specially  manufactured  by  the  body  cells  for  that 
purpose. 

The  term  antibody  is  used  to  designate  the  entire  group  of  spe- 
cific substances  produced  by  the  body  cells  in  reaction  against 
the  various  antigens;  certain  antibodies  act  by  neutralizing  their 
antigen  (antitoxin),  others  by  agglutination  or  precipitation  (agglu- 
tinins  or  precipitins) ;  others  act  by  completely  dissolving  their 
antigen  (bacteriolysins,  hemolysins) ;  still  others  may  so  lower 
the  resistance  of  their  antigen  as  to  render  it  an  easy  prey  to  the 
phagocytes  (opsonins  or  bacteriotropins) . 

Mechanism  of  Immunity.  —  Of  the  many  theories  advanced 
regarding  the  mechanism  of  immunity  two  have  claimed  more  at- 
tention than  all  the  others ;  one,  the  "  humoral  theory,"  advanced 
by  Ehrlich,  attributes  the  protective  forces  of  the  body  to  the  body 
fluids ;  the  other,  the  "  cellular  theory,"  proposed  by  Metchnikoff, 
claims  the  honor  for  certain  body  cells.  From  recent  studies, 
however,  it  is  evident  that  in  different  infections  the  body  employs 
different  means  of  protecting  itself.  In  certain  diseases  the  pro- 
duction of  immunity  seems  to  depend  upon  the  activity  of  the 
cells,  in  other  diseases  substances  contained  in  the  body  fluids 
seem  to  possess  the  property. 

Ehrlich's  Side  Chain  Theory.  —  The  "  humoral  theory  "  of 
immunity,  which  considers  that  the  power  of  resistance  to  infection 
resides  in  the  body  fluids,  is  said  to  have  originated  when  Foder 
discovered  that  the  blood  of  a  rabbit  when  placed  in  a  test  tube 
will  kill  anthrax  bacilli  without  the  apparent  aid  of  the  body  cells. 
Later,  in  1890,  additional  weight  was  given  to  the  theory  when  von 
Behring  and  Kitasato  demonstrated  the  presence  of  antitoxin 
in  the  blood.  At  first  great  importance  was  attached  to  the  anti- 
toxins, until  it  was  discovered  that  they  are  produced  only  in  a 
few  diseases,  notably  diphtheria,  tetanus,  and  botulism. 


IMMUNITY  125 

In  1894  Pfeiffer  discovered  that  cholera  spirilla  introduced  into 
the  peritoneal  cavity  of  a  guinea  pig  highly  immunized  against 
them  lost  their  motility  almost  immediately,  gradually  became 
swollen  and  granular,  and  finally  passed  into  complete  solution. 
These  changes  are  generally  spoken  of  as  "  Pfeiffer's  phenome- 
non," or  bacteriolysis. 

As  a  result  of  these  and  many  other  observations  Ehrlich  offered 
an  explanation  based  on  a  theory  advanced  some  years  before 
to  account  for  the  process  of  cell  nutrition.  He  thought  of  the 
cell  as  possessing  two  functions:  one  physiological,  such  as  that 
of  a  gland  cell  to  secrete,  and  the  second  function  one  of  nutrition 
concerned  mainly  in  nourishing  and  repairing  the  cell.  More- 
over he  attributed  this  double  function  to  each  molecule  com- 
posing the  complex  cell.  That  portion  of  the  molecule  by  means 
of  which  it  secures  nourishment  is  of  greatest  importance  in  rela- 
tion to  immunity.  Ehrlich  pictured  this  molecule  of  protoplasm 
as  a  functional  center  with  a  large  number  of  side  chains  or  recep- 
tions, each  of  these  side  chains  possessing  the  ability  to  select 
the  food  atom  it  needs  from  the  surrounding  blood  and  lymph, 
to  combine  with  it,  and  by  a  kind  of  absorptive  process  to  incorpo- 
rate it  in  the  molecule.  The  food  molecules  are  conceived  as  pos- 
sessing a  portion  specially  adapted  for  union  with  the  side  arm  of 
the  cell,  so  that  when  the  two  are  brought  in  relation  they  fit  to- 
gether in  much  the  same  way  that  a  key  fits  a  lock.  As  food  mole- 
cules vary  in  their  chemical  composition  it  is  reasonable  to  assume 
that  the  cell  receptors  prepared  to  anchor  them  differ :  they  may  be 
of  a  simple  constitution  and  adapted  only  to  the  taking  up  of  a 
very  simple  substance  or  they  may  be  more  complex  and  able  to 
digest  a  larger  food  molecule. 

It  is  easy,  then,  to  conceive  that  when  bacteria  and  other  foreign 
substances  are  introduced  into  the  body  fluids  certain  receptors 
may  anchor  them  as  they  pass  in  just  the  same  manner  as  a  simi- 
larly constituted  food  molecule.  Combination  with  such  sub- 
stances, however,  leads  to  a  different  result;  instead  of  being 
nourished  the  cell  may  be  poisoned  and  in  all  probability  lose  the 
receptor  that  had  attached  itself  to  the  foreign  substance.  If, 


126  BACTERIOLOGY  FOR  NURSES 

for  example,  that  substance  is  a  toxin  and  it  is  sufficiently  abundant 
and  powerful,  many  receptors  may  be  thus  lost  and  many  cells 
damaged .  Symptoms  of  the  specific  disease  may  present  themselves 
and  death  may  ensue.  On  the  other  hand,  if  the  injured  cells 
possess  sufficient  vitality  the  loss  of  one  or  more  receptors  will 
stimulate  them  to  an  immediate  attempt  to  repair  the  damage. 
Since  nature  is  always  lavish  in  her  processes  of  repair  it  may  hap- 
pen that  not  only  are  the  lost  receptors  replaced  but  a  large  num- 
ber are  added.  These  excess  receptors  having  no  place  for  attach- 
ment to  the  cell  are  thrown  off  into  the  blood  stream.  All  possess 
the  same  structure  as  the  original  ones.  Consequently  when  they 
meet  in  the  blood  stream  with  the  same  kind  of  substance  which 
caused  their  production,  their  antigen,  they  are  capable  of  com- 
bining with  it  and  rendering  it  harmless  before  it  is  able  to  attack 
the  body  cells.  In  diphtheria  and  tetanus  the  antigen  is  the  toxin 
of  the  bacteria,  and  the  cast-off  receptors  produced  as  a  result 
of  its  action  constitute  antitoxin. 

It  should  be  noted  that  an  excess  production  of  receptors  or 
antibodies  occurs  only  as  a  result  of  the  chemical  union  of  a  receptor 
with  a  poisonous  substance ;  the  assimilation  of  food  material  is 
of  benefit  to  the  cell ;  consequently  the  receptors  remain  unharmed. 

Three  Orders  of  Antibodies.  First  Order  (Antitoxins).  —  The 
simplest  receptor  of  the  cell  molecule  is  conceived  as  possessing 
a  single  arm  or  haptophore  for  union  with  a  correspondingly  simple 
food  molecule.  A  toxin  molecule  is  imagined  as  possessing  two 
portions,  one  the  haptophore  especially  adapted  to  fit  this  simple 
receptor,  and  a  second,  the  toxophore  portion  in  which  its  toxic 
power  resides.  The  cast-off  receptors  of  this  order  constitute 
antitoxin.  Fig.  21. 

Second  Order  (Agglutinins,  Precipitins) .  —  All  diseases  are 
not  produced  by  soluble  toxins,  and  furthermore  the  production  of 
antitoxin  does  not  explain  such  phenomena  as  the  agglutination 
of  bacteria  and  the  dissolving  property  of  certain  sera.  Accord- 
ingly Ehrlich  modified  his  theory  and  assumed  that  while  simple 
molecules  of  food  might  be  readily  assimilated  by  the  simple  cell 
receptor,  other  more  complex  molecules  might  require  some  form 


IMMUNITY 


127 


of  preparation  to  render  them  assimilable.  He  conceived  the 
possibility  then  of  a  second  order  of  receptors  furnished  like  the 
first  with  a  haptophore  portion  for  anchoring  the  food  molecule, 
and  also  with  an  additional  portion  which  he  called  the  zymophore 
group,  the  special  function  of  which  was  to  prepare  the  food  mole- 
cule for  absorption. 


FIG.  21.  —  Diagram  illustrating:  A,  Receptors  of  First  Order;  B,  Receptors  of 
Second  Order ;  C,  Receptors  of  Third  Order. 


, •'  Similarly,  certain  pathogenic  substances  which  are  more  com- 
plex than  the  toxins  he  assumed  would  combine  with  receptors 
of  this  kind,  the  haptophore  portion  of  the  pathogenic  molecule 
combining  with  the  haptophore  portion  of  the  receptor  and  acted 
upon  by  its  zymophore  portion. 

Two  such  antibodies  are  known ;  in  one  the  zymophore  por- 
tion of  the  antibody  causes  clumping  or  agglutination  of  its  antigen 
and  is  consequently  known  as  agglutinin.  In  typhoid  fever  the 


128  BACTERIOLOGY  FOR  NURSES 

bacilli  or  their  products  cause  the  production  of  an  antibody  of 
this  nature,  so  that  when  the  serum  of  a  typhoid  patient  is  mixed 
with  its  antigen,  typhoid  bacilli,  the  latter  lose  their  motility  and 
become  clumped  together  in  masses.  The  other  antibody  of  this 
class  appears  to  coagulate  and  precipitate  soluble  substances  and 
accordingly  is  known  as  precipitin.  Such  protein  substances  as 
milk,  egg  albumen,  etc.,  will  if  injected  into  an  animal  cause  the 
production  of  precipitin. 

Third  Order  (Bacteriolysins,  Hemolysins,  Cytolysins). — For 
still  more  complex  food  molecules  that  require  converting  into 
simpler  substance  before  they  can  be  assimilated  Ehrlich  con- 
ceived a  third  order  of  receptors  or  side  chains.  These  he  assumed 
to  be  composed  of  two  haptophore  or  grasping  portions,  one  for 
union  with  the  food  molecule  and  the  second  for  union  with  a 
special  ferment-like  substance  normally  present  in  the  blood 
and  called  by  him  "  complement " ;  by  French  writers  the  same 
substance  is  referred  to  as  "  alexin  "  or  "  cytase."  The  receptors 
therefore  act  simply  as  a  connecting  link  or  interbody  between 
the  food  and  the  complement,  serving  to  bring  them  in  relation 
with  each  other.  This  union  renders  the  food  molecule  soluble. 
In  other  words,  it  undergoes  lysis. 

Hemolysins,  bacteriolysins,  and  cytolysins  are  the  antibodies 
of  this  order.  If,  for  example,  the  red  blood  cells  of  one  animal 
are  injected  into  another  species  they  combine  with  the  receptors 
of  the  third  order  attached  to  the  cell.  As  they  are  toxic  the  result 
is  the  formation  of  antibodies  (hemolysins) .  If  more  of  the  anti- 
gen, which  in  this  case  is  the  foreign  corpuscles,  be  injected,  the 
hemolysins  uniting  them  with  the  complement  in  the  blood  cause 
them  to  be  completely  dissolved.  If  certain  bacteria  are  injected 
into  an  animal  immunized  against  that  particular  species,  conse- 
quently in  whose  blood  bacteriolysins  are  circulating,  they  share 
the  same  fate  as  the  corpuscles  in  the  previous  example.  Thus 
the  production  of  bacteriolysins  offers  a  satisfactory  explanation 
of  "  Pfeiffer's  phenomenon."  It  should  be  remembered  that 
although  these  antibodies  prepare  their  antigens  for  lysis,  or  "  sen- 
sitize" them,  they  are  not  in  themselves  lytic,  final  solution  of  the 


IMMUNITY  129 

antigen  being  accomplished  by  the  ferment-like  substance,  the 
complement. 

Ehrlich's  conception  of  immunity,  then,  was  that  of  a  chemist, 
and  his  antibodies  the  result  of  a  chemical  union  between  the  body 
cells  and  the  foreign  protein,  bacterial  or  other. 

"  The  Cellular  Theory."  —  Metchnikoff  as  a  biologist  naturally 
based  his  theory  on  his  observations  of  biological  phenomena. 
Since  all  cells  that  have  amebic  motion  are  capable  of  enveloping 
small  particles  he  considered  that  certain  body  cells,  and  partic- 
ularly the  leukocytes,  might  rid  the  body  of  infective  material 
and  thus  bring  about  a  state  of  immunity.  He  likened  these 
cells  to  scavengers  and  gave  to  them  the  name  of  phagocytes, 
because,  according  to  his  theory,  they  are  during  an  infection 
mainly  occupied  in  picking  up  and  disposing  of  offensive  material. 

He  divided  the  phagocytes  into  two  classes;  in  one  he  placed 
the  polynuclear  leukocytes  and  gave  them  the  name  of  micro- 
phages,  and  in  the  other  he  placed  the  endothelial  cells,  the  mono- 
nuclear  leukocytes,  and  embryonic  connective  tissue  cells.  To  these 
he  gave  the  name  macrophages.  The  former  play  an  active  role 
in  acute  pyogenic  infections,  the  latter  are  most  active  in  chronic 
bacterial  infections  such  as  tuberculosis  and  syphilis.  Metchnikoff 
soon  realized  that  phagocytosis  alone  could  not  explain  all  the 
phenomena  and  that  further  study  was  necessary.  He  found 
that  the  digestive  power  of  the  phagocytes  is  very  great,  and 
that  gradually  they  are  able  to  dissolve  almost  any  substance. 
He  considered  two  of  these  substances,  one  derived  from  the 
microphages  which  he  called  microcytose,  and  the  other  obtained 
from  the  macrophages  and  to  which  he  gave  the  name  of  macro- 
cytose,  to  be  identical  with  Ehrlich's  interbody  and  complement. 

The  relative  importance  of  the  cellular  and  humoral  theories 
rests  largely  on  which  of  the  body  cells  are  most  active  in  forming 
antibodies.  Metchnikoff  showed  the  important  part  played 
by  phagocytes  in  any  infection,  and  claimed  that  the  antibodies 
in  the  circulating  fluids  are  the  products  of  their  activity.  Ehrlich, 
on  the  other  hand,  emphasized  the  presence  of  immune  bodies 
in  the  body  fluids  and  sought  to  explain  the  method  by  which  they 


130  BACTERIOLOGY  FOR  NURSES 

are  produced  without  attributing  the  action  to  any  one  group  of 
cells. 

Recent  experiments  tend  to  show  that  the  leukocytes,  the  spleen, 
lymphatic  tissue,  and  bone  marrow  are  all  actively  concerned  in 
manufacturing  antibodies.  In  infections  due  to  the  various  patho- 
genic cocci,  phagocytes  appear  to  be  the  most  active  agents ;  in 
other  infections  such  as  those  due  to  typhoid  and  other  bacilli 
it  is  probable  that  antibodies  are  chiefly  operative  in  overcoming 
the  infection  and  producing  immunity.  Thus  there  appears  no 
warrant  for  holding  one  view  to  the  absolute  exclusion  of  the  other. 
It  would  seem  that  all  phases  of  immunity  are  cellular  in  origin 
and  that  this  cellular  activity  is  general  rather  than  limited  to 
one  group. 

Phagocytosis. ' —  In  order  to  understand  phagocytosis  it  is 
necessary  to  consider  its  three  phases ;  namely,  the  advance  of  the 
leukocytes,  the  engulfment  of  the  particles,  and  their  subsequent 
digestion.  The  question  naturally  arises  as  to  how  the  leukocytes 
are  made  aware  that  their  presence  is  needed  at  the  point  of  infec- 
tion ;  some  attractive  force  must  operate  between  this  point  and 
the  leukocytes  circulating  in  the  blood.  It  is  assumed  that  a  chem- 
ical substance  acts  as  an  attractive  force.  Almost  all  motile 
unicellular  organisms,  whether  animal  or  vegetable,  will  respond 
to  chemical  stimuli.  Generally  attraction  is  toward  a  given  point, 
constituting  what  is  known  as  positive  chemotaxis. 

Experiments  show  that  such  cell  movement  is  due  to  a  change 
in  surface  tension.  If  the  chemical  substances  which  cause  this 
apparent  stimulus  decrease  the  surface  tension  the  cell  advances 
in  the  direction  from  which  the  substance  comes,  if  it  decreases 
the  tension  the  cell  recedes. 

"  This  explanation  may  account  for  the  behavior  of  cells  in 
inflammation.  At  the  point  of  injury  or  infection  chemical  sub- 
stances are  produced  that  tend  to  lower  the  surface  tension; 
these  are  carried  by  the  body  fluids  to  the  nearest  capillaries 
where  they  enter  through  the  vessel  wall  and  come  in  contact  with 
the  leukocytes.  Naturally  the  surface  tension  will  be  least  on 
that  side  of  the  vessel  wall  through  which  the  stimuli  has  passed 


IMMUNITY  131 

and  the  result  will  be  the  forming  of  pseudopodia  on  the  part  of 
the  leukocytes  and  gradual  motion  in  this  direction.  Once  beyond 
the  vessel  wall  the  leukocyte  will  travel  in  the  direction  from  which 
the  chemotactic  substance  comes.  If  the  leukocyte  encounters 
a  substance  that  further  lowers  the  surface  tension  it  will  encircle 
and  inclose  it.  If  by  chance  it  has  engulfed  bacteria  their  toxins 
may  kill  the  cell  or  so  equalize  the  tension  that  it  ceases  to  move ; 
otherwise  the  leukocyte  moves  forward  until  it  is  checked.  It 
may  be  that  movement  continues  always  in  the  direction  from 
which  the  chemical  stimulus  comes  until  it  is  blocked  by  a  phalange 
of  leukocytes  which  are  being  held  by  chemotaxis  around  the 
area  of  infection.  This  supposition  would  explain  the  wall  forma- 
tion which  so  often  occurs  in  suppurative  processes.  If,  on  the 
other  hand,  recovery  commences  and  the  chemotactic  substances 
cease  to  be  formed,  then  it  may  be  more  abundant  at  some  dis- 
tance away  from  the  center,  and  towards  this  point  the  leukocytes 
will  move,  following  the  attracting  substance  back  to  the  lymph 
stream  and  blood  vessels.  This  probably  explains  the  dispersion 
of  the  living  phagocytes  at  the  end  of  an  inflammatory  process." 
(Kolmer.) 

Nothing  is  known  of  the  nature  of  these  chemotactic  substances. 
It  is  thought  that  they  may  be  formed  as  a  result  of  the  death  of 
tissue  cells  caused  by  bacterial  poison  or  such  irritants  as  coal 
or  stove  dust.  In  most  infections  chemotaxis  is  positive;  in  a 
few,  however,  and  particularly  those  caused  by  virulent  streptococci, 
the  phagocytes  do  not  appear  to  be  influenced  by  any  such  sub- 
stance. Whether  stimuli  are  ever  formed  by  bacteria  that  actually 
repel  leukocytes  is  not  as  yet  decided.  If  such  substances  do 
occur  they  must  closely  resemble  the  aggressins  which  neutralize 
opsonins  and  so  retard  phagocytosis. 

The  engulfment  of  bacteria  is  accomplished  by  the  phagocyte 
protruding  part  of  its  protoplasm  until  it  encircles  the  organism, 
which  soon  appears  within  the  substance  of  the  phagocyte. 

After  phagocytosis  the  fate  of  the  inclosed  bacteria  depends 
largely  on  their  nature.  Generally  they  undergo  a  process  of 
digestion  similar  to  gastric  digestion  in  higher  animals;  the 


132  BACTERIOLOGY   FOR  NURSES 

phagocytes  are  able  thus  to  dispose  of  particles  of  all  kinds,  bac- 
teria, cellular  debris,  the  fragments  of  coal  in  anthracosis,  catgut, 
and  silk  ligatures  and  other  foreign  substances.  It  may  happen, 
however,  that  they  can  withstand  the  phagocytic  ferments  and 
even  cause  the  death  of  the  cell,  in  which  case  they  become  again 
liberated  in  the  tissues.  The  inclosing  of  the  parasites,  then, 
is  only  a  preliminary  step.  The  process  may  be  useless  unless 
suitable  digestive  ferments  are  excreted  and  the  bacteria  dis- 
solved. 


CHAPTER  XIII 

OPSONINS,   AGGLUTININS,   PRECIPITINS,   LYSIN 

Opsonins.  —  The  process  of  phagocytosis  has  been  found  to 
depend  not  so  much  on  inherent  properties  of  the  phagocytes  as 
upon  a  certain  substance  present  in  the  body  fluids.  Apart  from 
this  substance  the  leukocytes  do  not  become  phagocytes.  Metch- 
nikoff  suggested  that  the  presence  of  this  substance  stimulated  the 
leukocytes  to  become  active.  Later  studies,  however,  have  shown 
that  the  substance  acts  directly  upon  the  bacteria  and  renders 
them  more  attractive  to  and  easily  digested  by  the  leukocytes. 
These  bacteriotropic  substances  have  been  named  opsonins  (from 
opsono,  I  prepare  food  for).  Opsonins  are  present  normally  in 
the  blood.  They  may,  however,  be  greatly  increased  in  immuni- 
zation. 

Bacteria  differ  in  their  susceptibility  to  opsonins.  Their  resist- 
ance may  be  due  to  capsule  formation  or,  according  to  the  theory 
of  Welch,  to  actual  self-immunization  of  the  bacteria.  Opsonins 
are  much  more  active  in  some  infections  than  in  others ;  they  are 
especially  operative  in  pyogenic  conditions,  in  which  phagocytosis 
is  recognized  as  the  chief  defensive  force.  The  relation  of  normal 
and  immune  opsonins  to  the  other  antibodies  is  as  yet  unsettled ; 
they  may  be  of  the  same  nature  as  amboceptor  and  complement 
or  they  may  be  separate  antibodies.  However  that  may  be,  they 
are  an  important  factor  in  the  production  of  immunity,  since  it  is 
upon  their  action  that  phagocytosis  depends. 

The  existence  of  opsonins  in  a  given  serum  is  easily  demonstrated 
by  mixing  the  serum  with  a  suspension  of  bacteria  and  adding 
washed  leukocytes;  the  leukocytes  will  in  all  probability  take 
up  large  numbers  of  the  bacteria.  If,  however,  leukocytes  washed 

133 


134  BACTERIOLOGY  FOR  NURSES 

free  from  serum  are  added  to  a  suspension  of  bacteria  which  have 
not  previously  been  sensitized  by  opsonins  practically  no  phagocy- 
tosis occurs. 

A  method  has  been  devised  whereby  the  relative  amount  of 
opsonins  present  in  the  blood  of  a  sick  person  and  a  normal  healthy 
person  may  be  estimated.  For  the  procedure  it  is  necessary 
to  have  (1)  blood  serum  from  the  patient  and  also  from  a  healthy 
person,  (2)  washed  leukocytes,  and  (3)  a  suspension  of  the  organ- 
isms the  opsonin  for  which  is  to  be  measured. 

Serum  is  obtained  from  the  patient  by  pricking  the  finger  or 
ear  lobe  and  allowing  the  blood  to  flow  into  a  Wright's  capillary 
tube  (Fig.  22).  The  tube  is  easily  made  by  bending  in  a  flame 
glass  tubing  with  a  small  lumen.  After  the 
clot  has  formed  the  tube  can  be  broken  and 


FIG.  22. —  A,  Wright's  Capillary  Tube  for  collecting  Blood ;  B,  Wright's 
Capillary  Pipette  used  for  Opsonic  Index. 

the  serum  withdrawn.  Serum  is  obtained  from  a  normal  indi- 
vidual in  the  same  manner. 

An  emulsion  of  leukocytes  is  prepared  by  dropping  about  twenty 
drops  of  blood  from  a  pricked  finger  into  20  c.c.  of  normal  saline. 
The  mixture  is  centrifuged  until  the  leukocytes  appear  as  a  layer 
of  cream  over  the  red  corpuscles.  The  clear  upper  fluid  is  removed 
by  means  of  a  pipette  and  the  upper  layer  of  the  sediment  added 
to  10  c.c.  of  normal  saline.  This  second  mixture  is  centrifuged, 
after  which  the  clear  upper  fluid  is  discarded  and  the  remaining 
leukocytic  emulsion  used  for  the  test.  The  purpose  of  this  prepara- 
tion is  to  wash  the  leukocytes  free  from  influencing  substances 
that  might  be  present  in  the  blood  from  which  they  were  taken, 
and  also  to  obtain  a  greater  number  of  leukocytes  in  a  small  quan- 
tity of  emulsion  than  would  be  found  in  the  same  amount  of  whole 
blood. 

The  bacterial  suspension  is  prepared  by  gently  rubbing  a  loopful 
of  the  growth  of  the  organism  taken  from  an  agar  culture  into 


OPSONINS,  AGGLUTININS,  PRECIPITINS,  LYSINS    135 

normal  salt  solution.  The  suspension  should  only  be  moderately 
thick  and  when  thoroughly  mixed  should  be  centrifuged  to  remove 
all  clumps. 

By  means  of  a  capillary  pipette  marked  about  one  inch  from 
the  end,  equal  amounts  of  the  three  fluids,  i.e.  patient's  serum, 
leukocytes,  and  bacterial  suspension,  are  drawn  up  into  the  tube, 
an  air  space  being  left  between  each.  All  are  then  expelled  upon 
a  slide  and  thoroughly  mixed  by  being  drawn  into  the  tube  and 
again  expelled  several  times.  Finally  the  mixture  is  drawn  into 
the  tube,  the  end  is  sealed,  and  the  tube  is  placed  in  an  incubator 
at  37°  C.  Exactly  the  same  procedure  is  repeated,  using  normal 
serum  in  place  of  the  patient's  serum.  After  fifteen  minutes' 
incubation  a  large  drop  of  the  mixture  from  the  tube  containing 
the  patient's  serum  is  smeared  on  a  slide  by  means  of  a  second 
slide  as  for  an  ordinary  blood  film.  A  second  slide  is  prepared  in 
the  same  manner  from  the  tube  containing  the  normal  serum, 
and  both  films  are  dried,  fixed,  and  stained.  The  slides  are  then 
examined.  The  number  of  bacteria  are  counted  in  fifty  or  one 
hundred  leukocytes  and  the  average  determined  by  dividing  the 
total  number  of  bacteria  counted  by  the  number  of  leukocytes  in 
which  they  were  found.  This  gives  the  phagocytic  index.  The 
opsonic  index  is  then  determined  by  dividing  the  phagocytic  index 
of  the  patient  by  that  of  the  healthy  individual.  Thus,  if  the 
average  number  of  bacilli  influenced  by  the  opsonin  in  the  patient's 
serum  and  taken  up  by  the  phagocytes  is  three,  and  the  average 
number  taken  up  by  the  leukocytes  after  being  acted  upon  by 
normal  blood  is  four,  then  the  opsonic  index  will  be  f  of  one  or  0.75. 
For  example : 

3=  patient's  phagocytic  index  _~  _. 
4=  normal  phagocytic  index 

Normally  the  opsonic  index  may  vary  between  0.8  and  1.2.  A 
higher  index  is  regarded  as  indicative  of  increased  resistance  and 
an  index  below  normal  as  diagnostic  of  infection  with  the  specific 
organism  tested.  In  the  above  case  the  patient's  index  is  below 
normal,  consequently  an  infection  might  be  assumed. 


136  BACTERIOLOGY  FOR  NURSES 

The  opsonic  index  as  a  guide  for  the  administration  of  vaccine 
was  at  one  time  considered  its  most  practical  use.  Observation 
showed  that  following  an  injection  of  vaccine  the  opsonins  were 
decreased  for  a  longer  or  shorter  period,  the  so-called  negative 
phase,  and  that  later  an  increase  occurred,  the  positive  phase. 
The  purpose  of  recording  the  opsonic  index  was  so  to  determine 
the  dose  and  the  time  of  administering  the  vaccine  that  the  nega- 
tive phase  should  be  as  limited  as  possible.  The  opsonic  index 
is  much  less  used  at  present  in  vaccine  therapy;  it  requires  a 
considerable  amount  of  time  to  determine  and  can  only  be  relied 
upon  when  undertaken  by  skilled  workers. 

Agglutinins.  —  According  to  Ehrlich's  side  chain  theory  agglu- 
tinins  are  antibodies  of  the  second  order.  They  possess  like 
antitoxins  and  lysins  a  portion  for  uniting  with  their  antigens. 
They  also  possess  zymophore  portions  to  which  their  special  reac- 
tion is  due.  Metchnikoff  believed  that  the  agglutinins  are  derived 
from  the  leukocytes  and  endothelial  cells.  It  is  generally  thought, 
however,  that  the  bone  marrow  and  the  spleen  are  most  active 
in  their  formation. 

If  the  serum  of  a  patient  suffering  from  typhoid  fever  is  added 
to  an  emulsion  of  typhoid  bacilli,  and  the  mixture  placed  in  the 
incubator  for  a  short  period,  the  bacteria  which  were  formerly 
scattered  throughout  the  fluid  will  be  found  to  have  clumped 
together  in  small  masses  at  the  sides  of  the  test  tube,  and  as  they 
gradually  fall  to  the  bottom  the  fluid  becomes  clear.  If  a  hang- 
ing drop  preparation  is  made  it  will  be  observed  that  with  the 
addition  of  serum  the  bacilli  move  closer  together,  gradually  losing 
their  motility,  until  finally  they  adhere  in  clumps. 

In  1896  Widal  applied  this  fact  to  the  diagnosis  of  typhoid 
fever,  and  since  agglutinins  appear  comparatively  early  in  the 
disease  it  is  a  considerable  aid  both  in  typhoid  fever  and  in  other 
diseases  in  which  agglutinins  are  formed.  The  test  may  be  made 
macroscopically  by  sedimentation  of  the  clumps  in  a  test  tube 
or  microscopically  in  a  hanging  drop ;  the  latter  method  is  best 
adapted  for  diagnostic  purposes  when  a  quick  report  is  necessary 
and  only  a  small  amount  of  serum  is  available. 


OPSONINS,  AGGLUTININS,  PRECIPITINS,  LYSINS     137 

Serum  may  be  obtained  by  pricking  the  finger  or  ear  lobe  with  a 
sharp-pointed  instrument  or  needle  and  allowing  the  blood  to  pass 
into  a  Wright's  capillary  tube,  as  already  described.  Whole  blood 
may  be  used,  in  which  case  one  or  two  drops  are  placed  on  a  slide 
and  dried  and  later  brought  into  solution  again  by  the  addition 
of  salt  solution.  The  use  of  serum  from  which  the  red  blood  cells 
have  been  removed  is  preferable. 

When  the  serum  has  been  obtained  a  1  in  10  dilution  is  made 
by  mixing  one  part  of  serum  with  nine  parts  of  normal  salt  solu- 
tion; from  this  dilution  higher  ones  are  made.  For  example, 
one  part  of  the  1  in  10  dilution  to  one  part  of  salt  solution  gives  a 
1  in  20  dilution ;  one  part  of  1  in  10  dilution  to  two  parts  of  salt 
solution  gives  a  1  in  30  dilution ;  etc. 

The  culture  with  which  the  test  is  to  be  made  should  be  either 
a  twelve  to  eighteen  hour  growth  in  broth  of  the  specific  organisms 
or  an  emulsion  of  an  eighteen  hour  agar  slant  culture  in  normal 
salt  solution.  The  latter  is  prepared  as  already  described  for 
an  opsonic  index. 

In  making  the  microscopic  test  the  procedure  is  much  the  same 
as  for  a  hanging  drop.  A  platinum  loopful  of  the  serum  dilution 
and  an  equal  quantity  of  the  bacterial  suspension  are  thoroughly 
mixed  on  a  coverslip,  and  the  coverslip  is  then  inverted  over  a 
concave  glass  slide  the  rim  of  which  has  been  greased  with  vaseline. 
Several  dilutions  are  used  and  the  slides  are  placed  in  the  incubator 
for  twenty  to  thirty  minutes  and  then  examined  with  the  number  7 
dry  lens.  In  a  series  of  dilutions  all  degrees  of  agglutination  may 
be  observed,  from  large  clumps  with  clear  interspaces  to  smaller 
clumps  with  a  few  motionless  bacteria  between,  down  to  clusters 
of  six  or  seven  organisms  partially  agglutinated  trying  to  free 
themselves.  In  a  positive  case  of  typhoid  fever,  within  thirty 
minutes  a  1  in  20  dilution  will  usually  show  complete  agglutina- 
tion, and  a  1  in  40  dilution  an  almost  complete  reaction ;  partial 
agglutination  may  sometimes  be  observed  in  a  dilution  as  high  as 
1  in  100.  Properly  killed  bacteria  respond  to  the  test  almost 
as  well  as  living  ones. 

The  macroscopic  test  is  made  as  follows :  a  series  of  small  tubes 


138  BACTERIOLOGY   FOR  NURSES 

are  arranged  in  a  rack  and  in  them  are  placed  equal  amounts 
of  serum  dilution  and  bacterial  suspension,  each  one  of  the  tubes 
receiving  a  dilution  of  serum  higher  than  the  preceding  one. 
It  should  be  remembered  that  the  addition  of  the  bacterial  sus- 
pension increases  the  serum  dilution;  thus  a  1  in  10  dilution  of 
serum  when  mixed  with  an  equal  volume  of  bacterial  suspension 
becomes  a  1  in  20  dilution. 

The  mixture  of  serum  and  bacteria  is  placed  in  the  incubator 
for  a  few  hours  and  then  left  at  room  temperature  or  placed  in 
the  ice  chest  for  several  hours.  When  agglutination  takes  place 
the  clumps  of  bacteria  fall  to  the  bottom  of  the  tube,  leaving  the 
fluid  entirely  or  partially  clear  according  to  the  amount  of  agglu- 
tinin  present  in  the  serum. 

In  typhoid  fever  a  positive  agglutination  reaction  may  be  given 
as  early  as  the  third  day  of  the  disease ;  ordinarily  it  does  not 
appear  before  the  seventh  or  eighth  day.  Occasionally  the  reac- 
tion may  be  absent  or  occur  only  during  convalescence ;  as  a  rule 
it  is  strongest  during  convalescence,  remains  positive  several 
weeks,  and  then  disappears. 

The  agglutinins  were  regarded  for  a  long  time  as  absolutely 
specific.  That  is,  that  dysentery  bacilli  were  agglutinated  by  serum 
of  an  individual  or  animal  immune  to  dysentery  and  by  no  other. 
Later  it  was  found  that  group  agglutinins  exist ;  that  is,  an  immune 
serum  will  agglutinate  closely  related  species  though  in  a  less 
degree.  For  instance,  the  serum  of  a  typhoid  patient  may  agglu- 
tinate typhoid  bacilli  in  a  1  in  80  dilution;  the  same  serum  may 
agglutinate  the  closely  related  colon  bacilli  in  a  1  in  10  dilution  and 
may  have  no  effect  whatever  on  the  unrelated  diphtheria  bacilli. 

In  addition  to  their  diagnostic  value  the  agglutinins  may  serve 
as  an  aid  in  the  differentiation  of  bacterial  species.  Thus  if 
serum  obtained  from  an  animal  highly  immunized  against  the 
typhoid  bacillus  agglutinate  an  unknown  organism  that  has  the 
cultural  characteristics  of  the  typhoid  bacillus,  the  unknown 
organism  is  undoubtedly  B.  typhosus.  Especially  may  this  be 
regarded  as  a  proof  if  the  unknown  strain  is  agglutinated  by  the 
same  dilution  of  the  serum  as  a  known  strain. 


OPSONINS,  AGGLUTININS,  PRECIPITINS,  LYSINS     139 

Precipitins.  —  Shortly  after  the  discovery  of  the  agglutinins  it 
was  shown  that  immune  serum  when  mixed  with  the  germ-free 
filtrate  of  a  culture  of  the  corresponding  organism  produced  a 
cloudiness  and  afterwards  a  precipitate.  Several  authorities  con- 
sider the  precipitins  and  agglutinins  are  identical.  It  is  reason- 
able to  assume  that  in  an  old  broth  culture  the  bacteria  undergo 
disintegration  and  pass  into  solution.  Accordingly  the  substances 
which  when  in  the  bacterial  body  are  agglutinated  may,  when  in 
solution  in  the  bacteria-free  filtrate,  be  precipitated.  The  test  is 
made  in  the  same  way  as  the  macroscopic  test  described  for  agglu- 
tinins, except  that  the  filtrate  of  a  culture  is  used  instead  of  a  bac- 
terial suspension. 

It  has  been  found  that  precipitins  may  be  produced  by  injecting 
albuminous  substances  into  suitable  animals.  Thus  a  rabbit  im- 
munized to  human  serum  will  produce  a  precipitate  when  mixed 
with  human  serum ;  similarly  a  rabbit  immunized  to  horse  serum 
will  produce  a  precipitate  when  mixed  with  horse  serum.  This 
fact  has  found  a  practical  application  in  forensic  medicine ;  blood 
spots  dissolved  out  in  normal  salt  solution  can  be  recognized  as 
of  human  or  animal  origin  even  after  months  of  drying.  The  test 
is  also  used  in  determining  the  nature  of  meat  suspected  to  be  horse 
flesh. 

Lysins.  —  The  bacteriolysins  were  discovered  by  Pfeiffer  in 
his  attempt  to  immunize  animals  against  cholera  by  the  injection 
of  live  cultures.  It  was  soon  discovered  that  the  lytic  action 
Pfeiffer  had  noticed,  the  so-called  Pfeiffer's  phenomenon,  would 
take  place  not  only  in  the  peritoneal  cavity  of  a  guinea  pig  but 
also  in  a  test  tube  when  the  immune  serum  of  the  animal  was  at 
once  mixed  with  its  antigen.  According  to  Ehrlich  this  disinte- 
gration of  bacteria  occurs  as  a  result  of  their  union  with  an  anti- 
body and  complement.  To  the  antibody  he  gave  the  name  of 
amboceptor  because  he  considered  it  as  an  interbody  linking  to- 
gether the  antigen  and  complement.  One  of  the  extraordinary 
facts  connected  with  the  reaction  is  that  the  active  part  of  the 
combination,  the  complement,  is  normally  present  in  the  blood, 
but  only  when  united  with  a  specific  amboceptor  can  it  affect  the 


140  BACTERIOLOGY  FOR  NURSES 

antigen.  Complement  is  a  delicate  substance,  destroyed  by  a 
moderate  temperature  (55°  C.),  and  disappears  from  serum  that 
is  kept  for  a  few  days.  Like  the  ferments  it  is  somewhat  unstable, 
but  it  differs  from  them  in  being  "  fixed  "  or  used  up  in  definite 
quantities. 

Bacteriolysins  are  produced  only  in  the  case  of  certain  organ- 
isms, and  of  these  the  typhoid  and  cholera  groups  are  the  most 
notable. 

Lysins  may  be  produced  by  antigens  other  than  bacteria.  If 
an  animal  be  injected  with  the  body  cells  of  another  species  it 
develops  antibodies,  cytolysins,  which  when  combined  with  comple- 
ment disintegrate  the  same  type  of  cells  that  were  employed  for 
their  production.  Cytolysins  have  been  obtained  with  leukocytes, 
kidney  cells,  and  other  organs  and  tissues.  When  the  cytolysins 
were  first  discovered  they  aroused  great  enthusiasm  in  the  hope 
that  it  might  be  possible  to  disintegrate  and  dissolve  such  foreign 
cells  as  cancer  and  other  tumors.  Unfortunately,  the  results 
have  been  disappointing ;  the  cytolysins  are  comparatively  weak 
and  not  very  specific. 

Hemolysins  have  perhaps  been  the  most  studied  of  all  the 
lysins,  owing  to  the  ease  with  which  the  reaction  may  be  observed. 
It  has  long  been  known  that  in  some  instances  the  blood  serum 
of  one  animal  has  the  power  in  a  certain  degree  of  dissolving  the 
red  blood  cells  of  an  animal  of  a  different  species.  Bordet  demon- 
strated that  if  an  animal  were  given  repeated  injections  of  the  red 
blood  corpuscles  of  another  species  the  serum  of  the  former  acquired 
the  property  of  dissolving  the  red  blood  cells  of  the  latter.  He 
found  also  that  this  property  disappeared  when  such  serum  was 
heated  at  55°  C.,  but  as  in  the  case  of  other  lytic  serum  it 
was  regained  when  fresh  serum  from  a  normal  animal  was 
added.  It  was  evident  that  the  immune  serum  contained  a  new 
substance,  the  amboceptor,  which  in  the  first  instance  had  united 
the  cells  with  complement  and  brought  about  their  destruc- 
tion, and  in  the  second  case  it  was  amply  demonstrated  that 
not  only  is  complement  destroyed  by  a  lower  temperature  than 
amboceptor,  and  that  it  is  indispensable  for  the  reaction,  but  also 


OPSONINS,  AGGLUTININS,  PRECIPITINS,  LYSINS     141 

that  the  complement  is  not  specific  but  is  present  in  normal 
blood. 

Bordet  and  Gengou  found  that  bacteria  or  red  blood  cells 
could  be  "  sensitized  "  by  placing  them  in  immune  serum  that  had 
been'  deprived  of  its  complement ;  that  is,  the  antigen  and  ambo- 
ceptor could  enter  into  a  loose  combination  without  the  former 
being  affected.  If,  now,  measured  amounts  of  fresh  serum  from 
a  non-treated  animal  be  added,  all  the  complement  contained  in 
it  is  "  fixed  "  or  absorbed,  with  the  result  that  these  sensitized 
bacteria  or  red  blood  cells  are  dissolved.  These  facts,  which 
emphasize  the  general  law  that  when  an  antibody  is  demonstrated 
it  may  be  assumed  that  the  antigen  is  or  has  been  present,  form 
the  basis  of  the  Wassermann  reaction  for  syphilis  and  the  comple- 
ment fixation  tests  for  gonococcus  infection,  streptococcus  infec- 
tion, etc. 

Thus  when  in  suitable  proportions  a  bacterial  antigen  (for 
example,  a  suspension  of  gonococci)  is  mixed  with  heated  serum 
containing  the  specific  amboceptor  (serum  of  a  patient  with  a 
gonococcic  infection),  and  fresh  serum  containing  complement 
(usually  that  of  a  guinea  pig)  is  added,  there  is  a  union  of  the 
antigen  and  amboceptor,  and  all  of  the  complement  is  absorbed. 
Since  there  is  no  visible  sign  that  such  a  union  has  taken  place, 
it  is  determined  by  adding  in  measured  amounts  an  emulsion  of 
red  blood  cells  together  with  their  specific  amboceptor  but  no  com- 
plement. The  red  blood  cells  will  remain  unchanged  because  there 
is  no  free  complement  left  to  unite  with  them. 

If,  on  the  other  hand,  the  suspected  patient  had  not  a  gonorrheal 
infection  and  consequently  no  specific  amboceptors  were  present 
in  his  serum,  then  the  complement  could  not  be  bound  to  the  anti- 
gen, so  that  on  the  addition  of  the  red  blood  cells  plus  their  ambo- 
ceptor the  complement  would  be  promptly  absorbed  by  them 
and  hemolysis  occur.  In  this  latter  case  a  marked  difference  in 
the  mixture  would  be  noted ;  the  disintegration  of  the  envelope 
of  the  cells  releasing  the  hemoglobin  would  give  to  the  fluid  a  clear 
red  appearance  totally  different  to  the  opaque  pinkish  color  of 
the  positive  case  in  which  the  red  blood  cells  remained  intact. 


142 


BACTERIOLOGY  FOR  NURSES 


The  reaction  may  be  represented  thus : 

(1)  Antigen+^^+Complement 
Red  blood  cells  -f-Hemoly tic  amboceptor 


(2) 


Red  blood  cells  +Hemoly  tic  amboceptor 


Positive  reaction ;  no  he- 
molysis,  the  complement 
having  combined  with 
the  antigen  and  specific 
amboceptor  in  patient's 


Negative  reaction ;  hemoly- 
sis  occurs.  Since  there  is 
no  amboceptor  in  the 

=  patient's  serum  the  com- 
plement was  free  to  unite 
with  the  red  blood  cells 
and  their  amboceptor. 


The  Wassermann  test  for  syphilis  has  the  same  principle,  save  that 
the  antigen  is  a  different  preparation. 

The  reaction  may  vary  all  the  way  from  a  complete  absorp- 
tion of  complement  to  a  non-absorption.  Complete  absorption 
of  a  given  amount  of  complement  is  regarded  as  strongly  positive 
and  is  frequently  reported  as  four  plus  (+  +  +  +)•  A  one  plus 
reaction  is  considered  as  only  doubtful  unless  recovery  is  taking 
place  as  a  result  of  medication. 

The  relation  between  the  various  antigens  and  their  antibodies 
may  be  tabulated  as  follows : 


ANTIGENS 

Soluble  toxins  (bacterial  or  other) 
Animal  and  vegetable  protein 
Bacteria 

Bacteria  and  red  blood  cells 
Bacteria  and  red  blood  cells  and  animal 
cells 


ANTIBODIES 

Antitoxins 
Precipitins 
Opsonins 
Agglutinins 

Bacteriolysins,  hemolysins, 
cytolysins 


CHAPTER  XIV 

TYPES  OF  ^IMMUNITY.    PREPARATION  OF  VACCINE. 
ANAPHYLAXIS 

RESISTANCE  to  bacterial  infection  may  be  inherent  or  natural 
or  it  may  be  acquired  as  a  result  of  an  attack  of  the  disease  or  by 
artificial  means.  Natural  immunity  is  an  inborn  quality  of  a 
species  and  of  course  is  the  converse  of  natural  susceptibility; 
acquired  immunity,  on  the  other  hand,  is  not  inborn  but  gained 
during  the  person's  lifetime.  It  is  a  state  of  natural  susceptibility 
transformed  into  one  of  resistance. 

Natural  Immunity.  —  This  type  of  immunity  is  an  inherited 
character  usually  possessed  by  all  individuals  of  a  given  species; 
thus  man  is  immune  to  certain  diseases  of  the  lower  animals, 
such  as  swine  plague,  fowl  cholera,  mouse  septicemia,  etc.  On  the 
other  hand,  animals  are  immune  to  many  of  the  diseases  common 
to  man,  such  for  example  as  measles,  typhoid  fever,  chicken  pox, 
etc. 

Closely  related  species  often  show  a  marked  difference  in  theii 
degree  of  resistance  to  the  same  infection ;  white  mice  are  practi- 
cally immune  to  glanders  whereas  field  mice  are  highly  susceptible. 
Negroes  are  said  to  be  more  susceptible  to  tuberculosis  and  less 
susceptible  to  yellow  fever  than  Caucasians.  In  man  the  differ- 
ence in  racial  immunity  is  not  so  marked  as  was  formerly  sup- 
posed ;  opportunities  for  infection  and  diverse  hygienic  customs 
may  in  a  large  measure  account  for  such  differences.  No  race  of 
mankind  seems  to  possess  absolute  immunity  to  any  disease  to 
which  the  species  is  susceptible. 

Individual  differences  are  often  noticed  in  the  degree  of  resist- 
ance to  infection  in,  for  example,  slight  cuts  and  scratches.  Indi- 

143 


144  BACTERIOLOGY  FOR  NURSES 

vidual  natural  immunity  is,  however,  a  more  or  less  relative  term  ; 
in  fact,  in  the  same  individual  slight  factors  such  as  exposure  to 
cold  or  fatigue  may  be  sufficient  to  change  the  balance  and  convert 
a  condition  of  resistance  into  one  of  susceptibility.  In  some  cases 
resistance  is  so  feeble  that  the  equilibrium  between  health  and 
disease  is  easily  disturbed ;  in  the  case  of  tuberculosis  in  man  the 
body  possesses  sufficient  natural  immunity  to  resist  small  amounts 
of  infection,  but  this  resistance  is  quickly  broken  down  by  any 
influence  which  undermines  the  general  vitality.  Hard  work, 
mental  and  physical,  which  involves  late  hours  and  inadequate 
periods  of  rest  and  recreation ;  insufficient  food  and  bad  air  —  all 
tend  to  lower  immunity  and  increase  susceptibility  to  infection. 
Exposure  to  wet  and  extreme  cold  is  well  known  as  a  factor  in  the 
etiology  of  colds  and  pneumonia.  Experiments  with  animals 
give  abundant  proof  of  these  facts.  For  instance,  chickens  ordi- 
narily immune  to  anthrax  may  become  susceptible  if  their  feet  are 
kept  in  cold  water ;  white  rats,  also  usually  immune  to  anthrax, 
become  susceptible  after  being  compelled  to  turn  a  revolving  wheel 
until  exhausted  before  they  are  inoculated. 

In  epidemics  of  certain  diseases  many  individuals  escape,  while 
in  other  persons  infection  appears  in  varying  degree  of  severity; 
It  is  probable  that  the  number  of  invading  organisms,  or  their  viru- 
lence, or  the  channel  of  infection  may  account  for  these  apparent 
differences  as  well  as  varying  degrees  of  immunity. 

Acquired  Immunity. — As  its  name  implies,  acquired  immunity  is 
the  converse  of  natural  immunity ;  it  is  acquired  and  not  inherent. 
Acquired  immunity  occurs  in  two  distinct  forms,  active  and  passive. 

Active  Acquired  Immunity  is  resistance  to  infection  due  to  the 
activity  of  the  body  cells  as  a  result  of  an  attack  of  the  disease  in 
question,  or  as  a  result  of  artificial  inoculation  with  the  specific 
organism  in  a  modified  form,  or  its  products.  Immunity  of 
this  kind  is  active  in  the  sense  that  it  occurs  as  the  result  of  the 
active  struggle  of  the  body  cells  against  the  invading  parasites, 
a  struggle  in  which  the  foe  is  overcome  and  the  body  cells  become 
more  resistant  than  they  were  before.  Active  immunity  may 
be  gained  by 


TYPES  OF  IMMUNITY  145 

(a)  An  attack  of  the  disease ; 

(6)  Introduction  of  vaccine  consisting  of  the  living  causal 
agent  in  a  modified  form ; 

(c)  Introduction  of  a  vaccine  consisting  of  dead  organisms ; 

(d)  Introduction  of  toxin. 

An  Attack  of  the  Disease.  —  The  degree  and  duration  of  immu- 
nity following  an  infection  varies  greatly  according  to  the  disease. 
Immunity  following  smallpox,  yellow  fever,  measles,  scarlet 
fever,  typhoid  fever,  whooping  cough,  typhus  fever,  chicken  pox, 
and  mumps  is  generally  lasting ;  in  a  few  of  the  diseases,  however, 
second  attacks  have  been  known  to  occur.  Certain  other  diseases, 
such  as  pneumonia  and  erysipelas,  seem  to  leave  the  individual 
more  susceptible  to  a  second  attack ;  yet  in  these  infections  there 
must  be  a  certain  amount  of  immunity,  even  though  it  is  of  short 
duration,  or  the  patient  would  not  recover. 

Introduction  of  the  Modified  Causal  Agent.  —  Apart  from  an 
actual  attack  of  the  disease  this  method  of  imitating  nature  pro- 
duces the  highest  and  most  lasting  degree  of  immunity.  Edward 
Jenner  in  1798  established  the  fact  when  he  succeeded  in  demon- 
strating that  vaccination  with  material  from  cowpox  protected 
an  individual  against  smallpox.  Eighty  years  later  Pasteur 
applied  Jenner's  principle  to  other  forms  of  disease.  About  1888 
the  chickens  in  the  neighborhood  of  Paris  were  being  destroyed  in 
great  numbers  by  a  virulent  intestinal  infection.  Pasteur  isolated 
an  organism  which  he  found  to  be  the  cause  of  the  disease  and  which 
when  injected  into  healthy  chickens  produced  all  the  characteris- 
tic symptoms.  Then  he  discovered  that  by  prolonged  cultivation 
on  artificial  medium  the  bacillus  could  be  so  attenuated  that  when 
injected  into  chickens  no  harm  resulted,  and,  what  was  of  much 
greater  importance,  these  same  chickens  when  inoculated  with 
freshly  isolated  virulent  organisms  were  found  to  be  immune. 

Pasteur  then  turned  his  attention  to  the  study  of  anthrax. 
He  found,  however,  that  the  methods  applied  to  immunize  chickens 
against  cholera  were  not  applicable  in  this  case.  Prolonged  culti- 
vation produced  spore  formation  and  not  attenuated  cultures. 


146  BACTERIOLOGY  FOR  NURSES 

Other  investigators  sought  to  obtain  the  desired  result  by  heating 
the  blood  of  animals  suffering  from  the  disease  for  a  few  minutes 
at  55°  C.  or  by  heating  cultures  of  the  organism  at  80°  C.  and 
then  using  them  for  inoculation.  Neither  of  the  methods  were 
very  successful.  After  much  experimentation  Pasteur  found  that 
by  cultivating  the  organisms  at  a  high  temperature,  42°  to  43°  C., 
they  could  be  so  attenuated  that  finally  they  were  entirely  robbed 
of  their  disease-producing  power.  In  this  manner  they  could  be 
modified  at  will,  and  by  inoculating  animals  first  with  a  highly 
attenuated  culture  and  then  with  a  moderately  attenuated  one 
he  was  able  to  immunize  them  against  anthrax. 

Immunization  against  hydrophobia  was  the  next  study  under- 
taken by  Pasteur,  and  here  as  in  the  two  previous  cases  his  efforts 
were  successful.  Again  he  was  confronted  with  a  difficulty  not 
met  with  before ;  in  this  case  the  causal  agent  was  unknown. 
First,  Pasteur  established  the  fact  that  the  virus  of  rabies  finds 
lodgment  in  the  brain  and  spinal  cord  since  by  injecting  an  emul- 
sion of  these  tissues  taken  from  an  infected  animal  into  rabbits 
he  was  able  to  reproduce  the  disease.  Then  he  discovered  that 
if  the  spinal  cords  were  removed  from  these  rabbits  and  subjected 
to  a  drying  process  the  virulence  of  the  virus  contained  in  them 
could  be  attenuated  to  whatever  degree  wished,  depending  upon 
the  length  of  the  period  of  drying. 

Pasteur  taught,  then,  at  least  three  methods  of  so  modi- 
fying organisms  that  they  may  be  used  for  the  artificial  pro- 
duction of  active  immunity :  (1)  prolonged  cultivation  on  media, 
(2)  growing  at  a  high  temperature,  and  (3)  drying.  He  also 
demonstrated  at  the  same  time  that  the  causal  agents  of  each 
disease  have  their  own  characteristics  and  must  be  dealt  with 
accordingly. 

In  certain  diseases  which  are  greatly  influenced  by  the  channel 
of  entrance  of  the  invading  organisms  still  another  method  is 
frequently  employed ;  the  most  resistant  tissues  are  chosen  as  the 
site  of  inoculation.  In  the  case  of  cholera,  for  example,  there  is 
much  less  danger  in  injecting  living  organisms  into  the  subcutane- 
ous tissues  than  in  taking  them  by  mouth. 


TYPES  OF   IMMUNITY  147 

Introduction  of  the  Dead  Causal  Agent.  —  This  method  is  of 
course  safer  than  the  preceding  one,  and  the  immunity  produced 
is  identical  with  that  produced  by  the  injection  of  living  organisms 
save  that  it  is  of  a  lower  degree  and  is  not  so  lasting. 

Vaccines  usually  produce  a  general  reaction,  such  as  malaise, 
headache,  pains  in  the  muscles,  and  slight  fever,  and  a  local  reac- 
tion at  the  point  of  inoculation.  The  reactions  appear  as  a  rule 
within  a  few  hours  and  last  from  one  to  two  days. 

A  vaccine  of  dead  organisms  is  prepared  from  a  twenty-four- 
hour  growth  of  a  pure  culture  on  an  agar  slant.  The  growth 
is  washed  off  with  salt  solution,  and  after  the  number  of  organisms 
present  in  the  suspension  are  determined  they  are  killed  by  being 
exposed  to  a  temperature  of  56°  C.  for  one  hour.  A  higher  temper- 
ature for  a  shorter  period  would  be  equally  effective  in  killing  the 
bacteria,  but  it  would  at  the  same  time  so  alter  the  organisms 
chemically  as  to  make  them  less  effective. 

Several  methods  are  in  use  for  determining  the  number  of  bac- 
teria present  in  such  a  suspension ;  the  following  is  one  of  the  most 
frequently  employed.  Blood  is  taken  from  a  pricked  finger  and 
a  blood  count  made  to  ascertain  the  number  of  red  blood  corpuscles 
present  in  a  cubic  millimeter.  Then  with  a  capillary  pipette 
one  volume  of  blood  is  taken  from  the  pricked  finger  and  mixed 
with  one  volume  of  the  bacterial  suspension  and  two  or  three 
volumes  of  sterile  salt  solution.  The  mixture  is  then  spread 
evenly  on  a  slide  as  in  making  a  blood  smear,  and  after  staining 
with  one  of  the  special  blood  stains  it  is  examined  with  the  oil 
immersion  lens.  The  red  blood  cells  and  the  bacteria  are  counted 
in  a  certain  number  of  fields  and  the  ratio  between  them  deter- 
mined. If,  for  example,  in  twenty  fields  the  average  shows  two 
red  blood  cells  to  one  microorganism  and  there  are  five  million 
red  blood  cells  in  each  cubic  millimeter  of  blood,  then  there  will  be 
approximately  half  that  number  of  bacteria,  or  two  million  five 
hundred  thousand,  in  a  cubic  millimeter  of  the  suspension  and  in 
a  cubic  centimeter  one  thousand  times  more.  A  vaccine  con- 
taining any  number  of  bacteria  desired  can  thus  be  obtained  by 
diluting  the  original  suspension.  A  simpler  method  is  that  of 


148  BACTERIOLOGY  FOR  NURSES 

centrifuging  the  suspension  in  a  special  tube.  A  sediment  of  or- 
ganisms up  to  a  certain  mark  gives  an  approximate  number  when 
diluted  with  a  given  quantity  of  salt  solution. 

Sensitized  Vaccines.  —  The  bacteria  living  or  dead  are  left  in 
contact  for  some  time  with  the  serum  of  an  animal  immunized 
against  that  particular  species  in  order  that  a  combining  of 
antigen  and  antibody  may  take  place,  after  which  the  serum  is 
removed  by  centrifuging.  It  is  claimed  that  immunity  produced 
by  sensitized  vaccines  is  more  quickly  developed  and  of  longer 
duration ;  also  the  local  and  general  reactions  are  lessened. 

Polyvalent  Vaccines.  —  Cultures  of  several  different  species  of 
bacteria  may  be  mixed  in  definite  proportions  and  administered 
at  the  same  time.  A  vaccine  containing  typhoid,  paratyphoid, 
A  and  B  bacilli,  and  cholera  spirilla  is  reported  to  have  been  used 
with  success. 

Bacterial  vaccines  are  always  given  in  subcutaneous  injections. 
Three  or  four  doses  are  usually  given  at  intervals  of  about  five 
to  ten  days. 

In  addition  to  being  used  as  a  prophylactic  measure,  vaccines 
are  often  employed  therapeutically  in  local  infections  such  as  acne, 
pustule,  or  a  boil.  It  is  assumed  that  while  the  local  resistance 
has  been  lowered  it  is  probable  that  the  general  antibody  produc- 
ing tissues  have  not  commenced  to  react ;  the  vaccine  may  thus 
stimulate  the  latter  and  cause  the  infected  area  to  be  flooded  with 
antibodies. 

Frequently  an  autogenous  vaccine  is  prepared  for  such  cases ; 
that  is,  the  infecting  organism  is  isolated  from  the  discharge, 
grown  in  pure  culture,  and  prepared  as  a  vaccine.  In  most  in- 
stances immunity  produced  by  the  introduction  of  a  bacterial 
vaccine  lasts  from  two  to  five  years  and  may,  of  course,  be 
renewed. 

It  is  not  definitely  known  how  long  a  vaccine  may  be  effectively 
used  after  the  date  of  its  preparation ;  usually  after  a  period  of 
from  four  to  six  months  it  is  supposed  to  lose  its  potency. 

Immunization  with  Toxin.  —  Soluble  toxin  such  as  produced 
by  the  diphtheria  bacillus  may  be  obtained  free  from  bacteria 


TYPES  OF  IMMUNITY  149 

by  filtration  of  a  broth  culture.  The  process  of  immunization  is 
started  by  giving  exceedingly  small  doses  usually  in  conjunction 
with  antitoxin;  afterwards  the  doses  are  gradually  increased. 
The  method  has  been  employed  in  the  case  of  snake  venom  and  a 
high  degree  of  immunity  thus  produced. 

Passive  Acquired  Immunity.  —  As  the  name  indicates,  this 
form  of  immunity  is  passively  acquired  by  virtue  of  receiving 
antibodies  formed  by  the  body  cells  of  an  animal  that  has  had 
to  resist  the  infecting  agent  in  order  to  produce  them.  Thus  in 
order  that  a  child  may  become  passively  immune  to  diphtheria 
an  animal  must  first  combat  the  disease ;  horses  are  injected  with 
successive  doses  of  toxin  and  are  required  to  overcome  its  effect 
and  acquire  an  active  immunity  of  a  high  grade  due  to  the  produc- 
tion of  antitoxin.  The  horse  then  is  actively  immune  because  it 
has  manufactured  its  own  antibodies.  When  its  antitoxin-laden 
serum  is  injected  into  the  child,  the  child  becomes  passively  im- 
mune ;  protection  being  due  not  to  the  activity  of  its  own  body 
cells  but  to  those  of  the  horse. 

Passive  immunity  is  specific ;  that  is,  the  serum  of  an  animal 
immunized  against  one  microorganism  will  protect  an  individual 
or  another  animal  against  that  and  against  no  other.  Immunity 
of  this  type  is  gained  just  as  soon  as  the  immune  serum  has  become 
mixed  with  the  blood  of  the  person  or  animal  injected.  It  is  of 
much  shorter  duration  than  active  immunity  and  the  degree  is 
seldom  equal  to  that  of  the  latter.  It  is,  however,  especially  of 
value  as  a  prophylactic  measure  against  an  acute  infection  that 
has  a  relatively  short  incubation  period. 

Ordinarily  the  injection  of  immune  serum  as  a  therapeutic 
measure  causes  very  little  disturbance  to  a  patient  and  this  little 
is  more  than  counterbalanced  by  the  release  of  the  body  cells 
from  combat  with  the  toxic  substances  which  are  overcome  by  the 
antibodies  injected. 

Passive  immunity  may  be  antitoxic  or  antibacterial,  according 
to  the  antigen  employed  for  the  production  of  the  specific  immune 
serum. 


150  BACTERIOLOGY  FOR  NURSES 

ANAPHYLAXIS 

Ordinarily  when  an  animal  is  given  repeated  injections  of  an 
antigenic  substance,  the  antibodies  produced  by  the  first  injection 
are  increased  and  eventually  a  high  degree  of  immunity  is  es- 
tablished. Under  certain  circumstances,  however,  the  reverse 
seems  to  be  the  case.  A  second  injection  will  produce  severe  and 
even  fatal  symptoms,  so  that  it  would  seem  instead  of  immunity 
a  state  of  hypersusceptibility  or  hypersensitivene.^s  has  been  pro- 
duced. 

To  this  state  of  hypersusceptibility  Richet  gave  the  name 
anaphylaxis,  meaning  "  without  protection,"  because  to  him  it 
represented  the  reverse  of  prophylaxis.  Recent  researches,  how- 
ever, tend  to  show  that  the  two  conditions  may  not  be  opposed, 
but  may  even  be  closely  related.  The  term  "  allergy  "  or  "  altered 
energy  "  has  been  suggested  as  a  more  appropriate  one. 

According  to  the  theory  of  Vaughan  the  phenomenon  of  ana- 
phylaxis may  be  explained  by  supposing  that  when  a  protein  such 
as  horse  serum  or  egg  albumin  or  bacteria  is  injected  it  is  broken 
up'by  an  enzyme  present  in  small  amounts  in  the  body  into  a  toxic 
and  a  non-toxic  portion.  At  the  first  injection  the  disintegra- 
tion takes  place  slowly  and  the  body  is  slightly  or  not  at  all  affected. 
By  the  second  time  the  injection  is  given,  however,  considerably 
larger  quantities  of  the  splitting  enzyme  have  been  elaborated, 
so  that  a  large  amount  of  the  toxic  portion  is  immediately  liberated 
and  symptoms  quickly  appear.  The  second  injection  causes  no 
symptoms  unless,  as  in  all  immunity  reactions,  sufficient  time  is 
allowed  to  elapse  for  the  cells  to  combine  with  the  proteins  and  for 
the  specific  ferments  to  be  produced.  The  exact  nature  of  the  re- 
action is  as  yet  unknown.  Recent  research  tends  rather  to  support 
the  view  that  anaphylaxis  occurs  as  a  result  of  the  union  between 
antigen  and  antibody,  taking  place  in  the  body  cells  and  not  in  the 
blood  stream.  Further  work  may  reveal  that  both  factors  are 
concerned. 

Experimental  anaphylaxis  in  animals  shows  that  the  first 
injection  of  a  foreign  protein  which  in  itself  is  not  poisonous  so 


ANAPHYLAXIS  151 

sensitizes  an  animal  that  a  second  injection  after  an  interval 
of  about  twelve  days  may  cause  the  reaction  known  as  anaphylac- 
tic  shock.  The  guinea  pig  is  apparently  the  most  susceptible  of 
all  animals  to  horse  serum,  yet  a  first  large  dose  gives  rise  to  no 
symptoms.  A  second  injection  of  a  minute  amount  may  cause, 
within  five  or  ten  minutes,  a  condition  of  restlessness  and  spasmodic 
respirations  and  probably  partial  or  complete  paralysis.  Recov- 
ery may  take  place  at  this  stage  or  convulsions  may  develop  and 
the  guinea  pig  may  die  within  twenty  or  thirty  minutes. 

The  symptoms  of  anaphylactic  shock  are  not  the  same  in  all 
animals,  and  this  has  been  explained  on  the  ground  of  slight  dif- 
ferences in  anatomical  structure.  It  has  been  demonstrated 
that  smooth  muscle  cells  are  the  most  hypersensitive.  In  the 
case  of  the  guinea  pig  the  mucosa  of  the  bronchi  is  relatively 
thick  compared  with  the  lumen  and  the  muscular  contraction 
throws  it  into  folds,  with  the  result  that  the  guinea  pig  is  asphyxi- 
ated. The  bronchi  of  dogs  have  relatively  less  smooth  muscular 
tissue.  This  probably  accounts  for  the  few  cases  of  death  from 
asphyxia  in  anaphylactic  dogs.  In  the  latter  contraction  of  the 
smooth  muscle  of  the  intestines  starts  a  vigorous  peristalsis ;  the 
muscles  of  the  heart  and  arteries  are  also  affected. 

Fortunately  the  severe  and  fatal  forms  of  anaphylaxis  are  ex- 
tremely rare  in  man ;  most  cases  have  occurred  in  persons  known 
to  be  susceptible  to  horse  protein.  This  undue  hypersusceptibility 
is  revealed  by  the  asthmatic  attacks  which  such  a  person  exhibits 
when  entering  a  stable  or  nearing  a  horse.  Serum  anaphylaxis 
or  "  serum  sickness  "  in  man  sometimes  occurs  following  a  dose 
of  antitoxic  serum ;  the  characteristic  symptoms  are  a  skin  erup- 
tion, swelling  of  the  lymph  glands,  joint  pains,  and  albuminuria. 
These  symptoms  are  altogether  independent  of  the  antitoxin  con- 
tained in  the  serum  and  are  purely  dependent  on  the  serum  as 
such.  For  this  reason  a  concentrated  antitoxin  is  less  likely  to  pro- 
duce serum  sickness.  In  the  majority  of  cases  symptoms  do  not 
appear  for  from  eight  to  ten  days.  Presumably  an  amount  of  the 
antibody  or  protein  splitting  substance  has  been  generated  by  that 
time  and  all  of  the  horse  serum  that  remains  in  the  circulation 


152  BACTERIOLOGY  FOR  NURSES 

is  attacked,  with  the  result  that  there  is  an  excess  of  liberated  poi- 
son which  gives  rise  to  the  anaphylactic  reaction.  If  the  dose  of 
serum  has  been  small  or  if  a  second  injection  follows  the  first  in 
less  than  six  to  eight  days  there  is  seldom  any  reaction.  If,  how- 
ever, a  second  injection  is  made  a  few  months  after  the  first  an 
immediate  reaction  often  follows.  If  a  year  or  more  elapse 
between  the  injections  there  is  usually  no  danger  of  a  reaction. 
By  that  time  the  antibody  has  disappeared  from  the  circula- 
tion. The  rare  fatal  cases  so  far  reported  have  followed  first 
injections,  presumably  because  the  individuals  were  already 
hypersensitive. 

Anaphylactic  or  allergic  skin  reactions  are  frequently  employed 
as  an  aid  in  diagnosis.  For  instance,  when  tubercle  protein  (tuber- 
culein),  syphilis  protein  (leutin),  glanders  protein  (mallein),  etc., 
is  applied  or  injected  into  the  skin  of  an  individual  sensitized  to 
that  particular  protein  a  local  reaction  occurs  characterized  by 
congestion  and  edema.  If,  for  example,  tuberculein  be  rubbed 
into  the  skin  of  a  normal  individual  he  will  not  react  because  he 
has  not  been  sensitized,  whereas  a  tuberculous  patient,  except  one 
in  the  last  stage,  reacts  promptly.  The  difference  between  the 
normal  individual  and  the  one  in  the  final  stage  of  tuberculosis 
is  that  the  former  has  not  been  sensitized  while  the  latter  has 
had  his  anaphylactic  powers  exhausted ;  consequently  he  presents 
little  or  no  resistance  against  the  advance  of  the  infection. 

The  so-called  food  idiosyncrasies  are  also  instances  of  anaphy- 
laxis.  The  articles  of  diet  usually  responsible  are  fish,  tomatoes, 
strawberries,  pork,  eggs,  etc. ;  the  symptoms  produced  are  skin 
eruptions,  gastro-intestinal  disorders,  and  vaso-motor  disturbances. 
When  there  is  a  difficulty  in  determining  which  food  is  responsible 
the  skin  test  may  be  employed  by  rubbing  a  drop  of  the  food  itself 
or  a  watery  extract  into  a  scratch  upon  the  skin.  The  reaction 
comes  on  within  thirty  minutes  and  is  demonstrated  by  a  pink 
red  edematous  area. 

The  Shick  skin  test  should  be  distinguished  from  the  above. 
It  is  not  an  allergic  skin  reaction,  it  depends  upon  an  entirely 
different  principle. 


PART  III 

CHAPTER  XV 

THE   PYOGENIC   COCCI 

THE  microorganisms  most  frequently  found  in  suppurative 
processes,  such  as  boils,  abscesses,  and  purulent  inflammations, 
belong  to  a  group  of  bacteria  known  as  pyogenic  cocci.  Of  these 
the  two  most  important  because  of  their  virulence  and  frequent 
occurrence  are  Staphylococcus  (pyogenes)  aureus  and  Streptococcus 
pyogenes. 

Early  investigators  noticed  the  frequent  presence  of  small 
round  bodies  in  the  pus  discharged  from  abscesses  and  sinuses 
and  gave  to  them  a  variety  of  names.  In  1880  staphylococci 
were  first  obtained  from  pus  by  Pasteur.  In  1881  Ogston  studied 
the  question  and  found  that  the  staphylococci  were  most  common 
in  circumscribed  acute  abscesses  and  the  streptococci  in  spreading 
suppurative  conditions.  Rosenbach  in  1884  differentiated  by 
means  of  cultures  several  different  varieties  of  pyogenic  micro- 
cocci  to  which  he  gave  the  special  names  staphylococcus  pyogenes 
aureus,  staphylococcus  pyogenes  aJbus,  streptococcus  pyogenes,  etc. 
Other  organisms  are  met  with  less  frequently  in  suppuration ; 
such  for  example  as  staphylococcus  pyogenes  citreus,  micrococcus 
tetragenus,  bacillus  pyocyaneus,  etc.  The  pyogenic  cocci  are  con- 
stant inhabitants  upon  the  skin  and  mucous  membranes.  Conse- 
quently not  only  may  they  cause  a  pathogenic  condition  themselves 
but  may  readily  enter  into  an  infection  started  by  another  organism 
and  further  increase  the  injury  by  giving  rise  to  a  "  mixed  infec- 
tion." 

153 


154 


BACTERIOLOGY   FOR  NURSES 


STAPHYLOCOCCUS  AUREUS 

Morphology  and  Staining.  —  The  organism  is  a  small  coccus 
about  0.8  fi.  in  diameter,  sometimes  appearing  in  pairs  or  isolated 
groups  of  three  or  four  but  most  commonly  in  irregular  clusters 
resembling  bunches  of  grapes.  It  stains  with  the  usual  basic 
anilin  dyes.  It  is  Gram  positive ;  that  is,  when  stained  by  the 
method  of  Gram  it  retains  the  gentian  violet  dye;  it  does  not 
form  spores ;  it  does  not  possess  flagella  and  is  consequently  non- 
motile,  although  marked  Brownian  movement  may  sometimes  be 

noticed    in     a    hanging   drop 
preparation.     (Fig.  23.) 

Cultivation.  The  organisms 
grow  readily  on  ordinary  arti- 
ficial culture  medium  made  of 
meat  extract.  The  optimum 
temperature  for  growth  is 
about  30°  C.  although  they 
possess  a  range  from  10°  C.  to 
43°  C.  in  which  multiplication 
will  occur.  In  stab  cultures  in 
peptone  gelatin  a  line  of  growth 
may  be  observed  the  day  after 
inoculation,  and  on  the  second  and  third  day  liquefaction  com- 
mences at  the  top  of  the  medium.  As  liquefaction  progresses 
the  growth  falls  to  the  bottom  as  a  flaky  deposit  of  a  golden 
yellow  color,  while  a  yellowish  film  may  remain  on  the  surface. 
In  gelatin  plates  colonies  appear  as  small  yellowish  disks  around 
which  liquefaction  soon  commences,  giving  a  cuplike  appearance 
with  the  small  colony  mass  at  the  bottom.  A  stroke  culture  on 
agar  gives  an  abundant  orange-yellow  growth  with  a  smooth 
shiny  surface.  Single  colonies  on  agar  appear  as  small  disks  of 
the  same  color.  On  potato  staphylococci  grow  luxuriantly  with 
an  abundant  production  of  pigment ;  in  broth  the  growth  appears 
as  a  turbidity  which  later  settles  to  the  bottom  as  a  sediment. 
Growth  is  rapid ;  it  is  estimated  that  a  broth  culture  may  contain 


FIG.  23.  —  Staphylococci. 


THE  PYOGENIC  COCCI  155 

in  twenty-four  hours  about  500,000,000  organisms  per  cubic 
centimeter.  They  are  able  to  ferment  dextrose,  lactose,  and 
saccharose,  and  from  them  form  various  acids.  Fermentation, 
however,  does  not  result  in  the  formation  of  gas.  Milk  is  coagu- 
lated and  indol  produced  in  peptone  solution  as  a  result  of  their 
growth. 

If  a  little  blood  is  added  to  nutrient  agar  and  staphylococci 
are  smeared  over  it,  a  clear  zone  surrounding  each  colony  appears 
after  twenty-four  to  thirty-six  hours'  growth;  this  effect  is  pro- 
duced by  a  hemolytic  substance  in  the  organism  which  dissolves 
the  envelope  of  the  red  blood  cells  and  sets  free  the  hemoglobin. 

Resistance.  —  Among  the  non-spore-bearing  bacteria  staphylo- 
cocci are  perhaps  the  most  resistant;  cultures  on  gelatin  or  agar 
will  remain  alive  for  a  year  or  more.  Suspended  in  water  the 
thermal  death  point  varies  with  different  cultures,  averaging  about 
two  hours  at  50°  C.,  one  half  hour  at  60°  C.,  and  ten  minutes  at 
70°  C.  They  are  killed  by  mercuric  chloride  1  in  1000  in  from 
fifteen  to  thirty  minutes,  and  by  carbolic  acid  1  in  100  in  from 
twenty  to  thirty  minutes. 

They  are  very  resistant  to  sunlight,  drying,  and  low  temperatures. 

Pathogenesis.  —  Animals  appear  to  be  considerably  less  sus- 
ceptible than  man  to  staphylococci  infections.  Large  amounts  of 
a  pure  culture  injected  into  a  rabbit  may  cause  the  formation  of 
abscesses  which  generally  heal  without  treatment;  or  if  the 
culture  is  sufficiently  virulent  and  a  large  enough  amount  be  given 
the  animal  may  die  in  from  two  to  eight  days.  On  autopsy, 
abscesses  are  found  in  the  various  internal  organs,  particularly 
the  liver,  kidneys,  and  in  the  walls  of  the  heart.  These  appear  as 
small  yellowish  masses  about  the  size  of  a  pea  surrounded  by  a 
zone  of  intense  congestion.  Many  of  the  capillaries  and  smaller 
arteries  are  blocked  with  thrombi  consisting  of  staphylococci. 

Investigators  have  produced  carbuncles  in  man  by  rubbing  a 
pure  culture  of  staphylococci  upon  the  unbroken  skin.  The 
organisms  supposedly  gain  entrance  into  the  deeper  tissues  through 
the  base  of  the  hair  follicles  or  sweat  ducts.  Lowered  vitality  of 
the  tissues  in  almost  any  locality  may  permit  a  local  invasion 


156  BACTERIOLOGY  FOR  NURSES 

resulting  in  a  boil  or  a  carbuncle.  Endocarditis,  septicemia,  or 
pyemia  may  result  from  a  local  abscess  through  the  introduction 
of  the  organisms  into  the  lymph  or  blood  stream.  Bone  tissue 
seems  to  be  particularly  susceptible.  The  majority  of  all  the 
attacks  of  osteomyelitis  and  periostitis  are  due  to  staphylococcic 
infection. 

Two  substances  have  been  isolated  from  cultures  of  staphylo- 
cocci  which  explain  in  part  their  ability  to  produce  disease.  One, 
staphylolysin,  acts  on  the  envelope  of  the  red  blood  cells  in  such 
a  manner  as  to  dissolve  out  the  hemoglobin,  and  is  consequently 
responsible  in  part  for  the  anemia  present  in  such  infections. 
The  other  substance,  leukocidin,  has  an  injurious  effect  upon 
leukocytes.  Both  of  these  substances  resemble  the  true  toxins 
in  that  they  stimulate  the  body  cells  to  produce  neutralizing 
antibodies.  It  is  more  than  probable  that  these  organisms 
generate  other  toxic  bodies,  but  as  yet  nothing  definite  is  known 
about  them.  Dead  culture  of  staphylococci  when  injected  sub- 
cutaneously  may  produce  local  abscesses. 

Immunity.  —  Phagocytosis  in  this  case  is,  without  question, 
the  chief  factor  in  immunization ;  the  amount  of  opsonin  is  in- 
creased, positive  chemotaxis  occurs,  and  the  phagocytes  actively 
engage  in  carrying  off  the  invaders.  An  immune  animal  serum 
has  been  prepared  containing  antibodies  that  neutralize  staphy- 
lolysin and  leukocidin.  Its  effect,  however,  is  relatively  weak 
and  it  is  seldom  used  except  to  confer  passive  immunity  in  cases 
in  which  the  general  vitality  is  so  low  as  to  contraindicate  the 
attempt  to  produce  active  immunity  by  the  introduction  of 
vaccine.  As  a  therapeutic  measure  vaccine  treatment  has  given 
most  satisfactory  results,  doses  commencing  with  2  to  20  million 
organisms  gradually  increasing  to  100  to  1000  million  are  given. 

Staphylococcus  pyogenes  albus.  This  coccus  is  identical  with 
staphylococcus  aureus  except  that  it  does  not  produce  a  yellow 
pigment  and  its  pathogenic  powers  are  somewhat  feebler.  Surface 
cultures  have  a  milk-white  appearance.  It  has  been  suggested 
that  it  may  be  a  degenerate  descendant  of  aureus,  but  no  one  has 
succeeded  as  yet  in  transforming  one  form  into  the  other. 


THE  PYOGENIC  COCCI  157 

Staphylococcus  epidermidis  albus.  It  is  probable  that  this 
organism  is  identical  with  Staphylococcus  albus.  It  is  slightly 
virulent  and  is  frequently  found  in  the  upper  layers  of  the  epi- 
dermis ;  it  is  the  common  cause  of  "  stitch  abscesses  "  following  a 
surgical  operation. 

Staphylococcus  pyogenes  citreus.  This  organism  also  differs 
from  aureus  only  in  the  color  of  its  pigment,  which  is  usually  of  a 
bright  lemon  yellow.  It  is  less  often  met  with  in  wounds,  however, 
than  either  of  the  preceding  cocci.  A  number  of  other  staphylo- 
cocci  exist,  few  of  which  are  in  any  degree  pathogenic  so  far  as 
is  known.  They  differ  in  minor  details,  such  as  ability  to  liquefy 
gelatin  or  to  form  pigment. 


MICROCOCCUS  TETRAGENUS 

In  1887  Gaffky  isolated  the  organism  from  the  pus  of  a  tubercu- 
lous patient.  It  has  been  observed  associated  with  other  organ- 
isms in  pulmonary  tuberculosis,  in  acute  abscesses,  and  also  in 
the  pus  of  empyema  following  pneumonia.  It  is  also  frequently 
found  in  the  saliva  of  healthy  persons,  and  it  is  generally  assumed 
that  while  it  rarely  incites  disease  its  presence  helps  to  contribute 
to  the  progressive  destruction  of  tissue  in  diseased  conditions. 

Morphology  and  Staining.  —  The  cocci  are  somewhat  larger 
than  staphylococci ;  their  diameter  averages  about  1  micron.  They 
are  arranged  regularly  in  groups  of  four  or  tetrads,  and  often  when 
first  removed  from  pus  appear  to  be  surrounded  by  a  capsule. 
They  are  readily  stained  by  the  basic  anilin  dye  and  are  Gram 
positive. 

Cultivation. — The  optimum  temperature  is  from  35°  C.  to 
38°  C.  Growth  is  slow,  but  will  occur  both  in  the  presence  and 
absence  of  oxygen.  On  agar  the  colonies  appear  as  small,  round 
points,  at  first  transparent  and  later  becoming  a  grayish  white. 
Gelatin  is  not  liquefied ;  acid  and  coagulation  is  produced  in  milk. 

Pathogenesis.  White  mice  are  especially  susceptible  to  in- 
fection by  the  micrococcus  tetragenus ;  other  animals  are  much 
less  so.  In  man  it  is  usually  non-pathogenic  except  in  the  condi- 


158 


BACTERIOLOGY  FOR  NURSES 


tions  already  referred  to ;  a  few  rare  cases  are  on  record  in  which 
it  has  been  cited  as  the  sole  producer  of  a  pyogenic  condition. 


STREPTOCOCCI 

The  pathogenic  streptococci  were  first  discovered  by  Koch  in 
stained  sections  of  diseased  tissue,  and  by  Ogston  in  1881  in  the 
pus  of  acute  abscesses.  Later  in  1883  Fehleisen  obtained  pure 
cultures  from  a  case  of  erysipelas.  Because  of  the  variety  of 
pathologic  conditions  in  which  streptococci  were  found  it  was  at 
first  thought  that  each  was  produced  by  a  different  species ;  now 
it  is  generally  assumed  that  the  slight  differences  between  the 

streptococci  of  erysipelas,1  of 
acute  abscesses,  of  septicemia, 
of  puerperal  fever,  etc.,  are 
only  acquired  variations  of 
organisms  of  the  same  spe- 
cies. 

All  spherical  bacteria  which 
divide  in  one  plane  only  and 
remain  attached  in  longer  or 
shorter  chains,  resembling 
somewhat  a  string  of  beads, 
are  classified  together  under 
the  name  of  streptococci  (Fig. 
42).  The  classification  is  simply  a  morphological  one  and  includes 
both  saprophytic  and  parasitic  varieties.  The  relationship  be- 
tween the  streptococci  from  different  sources  is  by  no  means  clear. 
Those  of  greatest  importance,  however,  because  of  the  power 
which  they  possess  of  inciting  disease  in  man  and  because  such 
diseases  are  frequently  of  a  suppurative  character,  are  roughly 
grouped  together  as  streptococcus  pyogenes. 

Streptococcus  pyogenes.  Morphology  and  Staining. — The  cocci 
are  relatively  small,  measuring  from  0.5  micron  to  1  micron  in 
diameter ;  they  have  no  flagella  nor  do  they  produce  spores.  As  a 
rule  the  pathogenic  streptococci  show  a  tendency  to  remain  united 


FIQ.  24.  —  Streptococci. 


THE  PYOGENIC  COCCI  159 

in  long  chains.  A  differentiation,  however,  cannot  be  based  on  this 
feature,  since  cultivation  on  media  relatively  unsuitable  causes  it  to 
disappear.  All  streptococci  are  stained  by  the  usual  anilin  dyes ; 
the  pyogenic  group  are  for  the  most  part  Gram  positive. 

Cultivation.  —  The  most  favorable  temperature  for  their  growth 
is  from  30°  C.  to  37°  C. ;  below  15°  C.  and  above  43°  C.  growth 
rarely  takes  place.  They  are  faultative  anaerobes  growing  both 
in  the  presence  and  absence  of  oxygen.  Upon  ordinary  nutrient 
agar  their  growth  is  rather  scanty.  Much  more  satisfactory  results 
are  obtained  by  cultivating  them  on  media  having  meat  infusion 
as  a  basis  or  on  media  to  which  blood,  serum,  or  ascitic  fluid  has 
been  added.  One  to  two  per  cent  of  glucose  added  to  the  medium 
favors  development  also.  In  a  gelatin  stab  growth  is  seen  about 
the  second  day  as  a  thin  line,  later  it  appears  to  be  formed  of  a 
row  of  minute  rounded  whitish  colonies;  the  growth  does  not 
spread  on  the  surface  and  no  liquefaction  occurs.  On  agar  or 
gelatin  surface  colonies  appear  as  fine  grayish,  opalescent  points, 
smooth  and  round,  or,  as  seen  under  the  low-power  lens,  with  a 
lacelike  edge  composed  of  chains  of  streptococci  arranged  in  loops. 
Acid  is  produced  in  milk  and  usually  coagulation  of  the  casein. 
In  slightly  alkaline  broth  after  twenty-four  to  forty-eight  hours 
the  growth  frequently  appears  as  a  deposit  of  tiny  flakes.  The 
addition  of  blood  or  serum  to  the  broth  seems  to  have  a  marked 
effect  on  their  chain  formation ;  the  same  strain  will  often  remain 
attached  in  long  chains  in  the  latter  case,  whereas  in  ordinary 
broth  they  may  separate  in  twos  or  threes.  On  Loeffler's  blood 
serum  medium  growth  is  rapid  and  luxuriant. 

The  pyogenic  streptococci  have  been  divided  into  two  groups 
on  the  basis  of  their  ability  to  cause  hemolysis.  If  blood  agar 
plates  are  prepared  by  adding  1  c.c.  of  fresh  or  defibrinated  blood 
to  6  c.c.  of  agar  at  43°  C.  and  then  inoculated  with  hemolytic 
streptococci  and  incubated  for  twenty-four  hours,  each  of  the 
colonies  will  appear  to  be  surrounded  with  a  clear  zone  due  to  the 
destruction  of  the  red  blood  cells.  Related  streptococci,  on  the 
other  hand,  produce  a  zone  of  a  greenish  color.  The  latter  are  not 
so  virulent  and  cause  rather  a  chronic  form  of  inflammation. 


160  BACTERIOLOGY  FOR  NURSES 

The  inulin  serum  medium  of  Hiss  is  frequently  used  to  differen- 
tiate the  streptococcus  pyogenes  from  pneumococci.  The  latter 
produce  acid  and  coagulation  of  the  serum,  while  the  former  are 
unable  to  ferment  inulin. 

Resistance.  —  On  culture  media,  unless  transplanted,  strepto- 
cocci do  not  live  more  than  two  to  fourteen  days ;  in  body  dis- 
charge they  may  live  for  several  weeks.  They  are  killed  by 
exposure  to  a  temperature  of  54°  C.  for  twenty  minutes;  low 
temperatures  have  less  effect  upon  them.  Exposure  to  sunlight 
kills  them  in  a  few  hours.  Mercuric  chloride  1  to  1000  destroys 
them  in  from  five  to  ten  minutes,  carbolic  acid  1  to  100  in  from 
five  to  forty-five  minutes. 

Pathogenesis.  —  Attempts  have  been  made  to  classify  the 
streptococci  according  to  their  pathogenicity  or  their  growth  on 
artificial  culture  media;  such  attempts  have  not  been  very  suc- 
cessful because  the  differences  observed  are  not  constant.  What 
may  have  been  thought  to  be  a  definite  characteristic  may  become 
totally  changed  under  other  conditions.  The  animals  ordinarily 
used  for  experimentation  are  not  so  susceptible  to  streptococci 
infection  as  man,  and  different  animals  show  different  degrees  of 
susceptibility  to  different  cultures.  A  virulent  strain  when  in- 
jected into  a  mouse  will  cause  septicemia;  those  of  a  little  less 
virulence  will  produce  the  same  result  if  the  quantity  is  increased ; 
others  still  less  virulent  will  produce  septicemia  if  injected  into  a 
vein,  but  if  introduced  into  subcutaneous  tissue  will  produce  an 
abscess  or  erysipelas.  Others  even  less  virulent  when  injected  in 
large  amounts  will  only  produce  a  slight  inflammation  or  no  re- 
action at  all. 

Experiments  have  shown  that  streptococci  originally  virulent 
may  become  non-virulent  after  long  cultivation  on  artificial 
culture  media,  but  that  after  passage  through  an  animal  they 
regain  their  lost  power. 

In  man  streptococci  are  responsible  for  a  greater  variety  of 
lesions  than  any  other  microbes,  and  in  addition  to  the  number 
of  diseases  they  themselves  cause  they  are  present  in  "  secondary  " 
or  "  mixed  "  infection  more  often  than  any  other  organisms. 


THE  PYOGENIC  COCCI  161 

Erysipelas,  a  spreading  inflammatory  condition  of  the  skin,  is 
almost  invariably  due  to  streptococci.  The  organisms  are  found 
in  large  numbers  in  the  underlying  tissues  and  lymphatics;  they 
may  extend  to  serous  and  synovial  cavities  and  give  rise  to  peri- 
tonitis, meningitis,  and  synovitis.  As  a  rule  in  erysipelas  the 
cocci  are  not  present  in  the  central  portion  of  the  inflamed  area, 
but  may  be  isolated  from  the  swollen  edge  by  excising  a  small 
piece  of  the  skin. 

The  fact  that  puerperal  fever  might  be  caused  by  infection 
from  an  erysipelas  case  was  noticed  long  before  it  was  discovered 
that  the  same  organism  could  produce  both  conditions. 

Observers  have  noted  that  patients  suffering  from  malignant 
tumor  seem  to  improve,  and  in  some  cases  the  tumor  has  diminished 
after  an  attack  of  erysipelas.  Fehleisen,  accordingly,  inoculated 
hospital  patients  suffering  from  inoperable  growths  with  cultures 
of  streptococci  and  produced  in  them  typical  erysipelas  and  often 
a  favorable  influence  on  the  growth.  Later  Coley  modified  the 
treatment  by  using  a  mixture  of  dead  streptococci  and  bacillus 
prodigiosus  or  their  products.  The  sarcomatous  tumors  are  most 
favorably  affected  by  "  Coley's  mixture  "  ;  carcinomatous  growths 
slightly  or  not  at  all.  Many  observers,  however,  have  failed  to 
note  any  favorable  results  following  its  use. 

Suppurative  conditions  in  different  organs  of  the  body  may 
result  from  streptococcus  invasion.  Ulcerative  endocarditis, 
bronchopneumonia,  pleurisy,  empyema,  otitis  media,  enteritis, 
are  included  in  the  list  of  diseases  of  which  they  may  be  the  primary 
cause. 

In  throat  affections  of  all  kinds  they  play  an  active  role ;  their 
constant  presence  on  the  mucous  membranes  and  tonsils  make 
possible  a  speedy  invasion  whenever  there  is  a  lowering  of  the 
local  vitality. 

In  smallpox  and  scarlet  fever  streptococci  can  be  isolated  from 
the  internal  organs  in  a  large  number  of  the  fatal  cases.  Certain 
authorities  regard  the  streptococcus  as  the  causal  agent  of  scarlet 
fever ;  the  view,  however,  is  not  generally  accepted. 

Isolated  from  the  blood  of  rheumatic  fever  cases  they  have  pro- 
M 


162  BACTERIOLOGY  FOR  NURSES 

duced  when  inoculated  into  rabbits  characteristic  arthritic  lesions. 
The  question  as  to  the  specificity  of  the  streptococcus  found  in 
rheumatism  is  as  yet  unsettled.  In  certain  cases  of  chronic 
arthritis  hemolytic  streptococci  have  been  isolated  from  the 
tonsils  which  when  injected  into  certain  animals  have  invariably 
produced  arthritis.  Removal  of  the  tonsils  in  such  cases  generally 
results  in  marked  improvement  or  recovery. 

Several  epidemics  of  sore  throat  due  to  streptococci  have  arisen 
from  time  to  time,  many  of  which  have  been  traced  directly  to 
the  milk  supply.  It  is  thought  that  such  streptococci  come 
originally  from  a  septic  human  throat,  find  their  way  into  the 
milk  ducts,  probably  from  the  hands  of  a  milker,  and  multiply 
there  without  causing  any  perceptible  inflammatory  condition 
in  the  eow.  The  bovine  type  of  streptococci  which  produce 
inflammations  of  the  udder  of  the  cow  has  different  cultural 
characteristics  and  is  apparently  not  identical  with  those  found 
in  human  septic  sore  throat. 

As  already  stated,  a  satisfactory  classification  of  streptococci 
from  different  sources  is  extremely  difficult.  Association  with  a 
specific  pathologic,  condition  is  not  conclusive  that  the  organism 
is  the  causal  agent  of  that  and  of  no  other  condition.  A  strain 
isolated  from  suppurative  processes  may  produce  erysipelas,  and 
conversely  abscess  formation  may  be  produced  by  one  isolated 
from  erysipelatous  lesions.  Classification  according  to  aggluti- 
nating reactions  or  the  ability  to  ferment  different  sugars  have 
likewise  given  insufficient  aid  in  determining  whether  the  strepto- 
cocci are  one  or  several  species. 

Immunity.  —  Streptococcus  inflammations  apparently  do  not 
stimulate  the  human  body  cells  to  produce  immunizing  anti- 
bodies. It  is  true  that  some  protective  substances  must  be  pro- 
duced or  recovery  would  not  take  place.  Evidently,  however,  they 
soon  disappear,  leaving  the  individual  as  susceptible  as  before. 

An  interesting  experiment  was  tried  by  Koch  and  Petruschky 
on  a  man  suffering  from  a  malignant  growth.  They  inoculated 
him  subcutaneously  with  streptococci  obtained  from  a  case  of 
erysipelas  and  produced  in  him  a  moderately  severe  attack  of 


THE  PYOGENIC   COCCI  163 

the  disease.  The  symptoms  disappeared  after  about  ten  days 
and  they  then  reinoculated  him  over  the  same  area  and  again 
obtained  the  same  result.  Ten  successive  attacks  were  produced 
in  the  same  manner,  which  proved,  at  least,  that  immunizing 
substances  were  not  present  in  sufficient  amounts  to  afford  pro- 
tection. It  is  a  well-established  fact  that  opsonin  is  increased 
and  phagocytosis  consequently  active  in  all  infections  with  the 
pyogenic  cocci. 

A  degree  of  active  immunity  may  be  produced  in  rabbits  and 
horses  by  inoculating  them  with  gradually  increasing  doses  of 
streptococcus  cultures.  Experiments  with  animals  have  shown 
that  the  serum  from  such  immune  animals  will  protect  to  a  cer- 
tain extent  against  the  organism  used  for  its  production,  but  not 
against  other  strains. 

Accordingly,  in  order  that  the  serum  may  contain  antibodies  to 
combat  the  different  streptococcic  infections,  a  number  of  different 
strains  isolated  from  different  forms  of  disease  are  used  for  inocu- 
lating the  horses.  The  "  polyvalent  "  serum  obtained  from  ani- 
mals so  treated  is  not  so  efficient  as  one  prepared  from  the  organisms 
infecting  the  treated  case,  but  it  is  moderately  effective  for  all  cases. 
The  use  of  such  serum  seems  to  have  been  of  benefit  in  certain 
cases,  but  on  the  whole  the  results  have  been  disappointing.  Large 
doses  must  be  given  in  order  to  obtain  a  sufficient  amount  of  anti- 
bodies to  produce  an  appreciable  effect. 

Vaccines  are  administered  in  subacute  conditions.  The  initial 
dose  is  5  to  10  million  organisms;  increasing  amounts  may  be 
given  to  500  million  as  a  maximum. 


CHAPTER  XVI 


PNEUMOCOCCUS,   MENINGOCOCCUS,   GONOCOCCUS 

THE  term  pneumonia  is  used  to  designate  a  variety  of  pathogenic 
conditions  of  the  lung  or  the  parts  which  compose  it.  Of  these 
the  two  forms  most  generally  met  with  are :  lobar  pneumonia 
(acute  croupous),  an  inflammatory  process  accompanied  by 
abundant  fibrinous  exudate  rapidly  involving  the  entire  tissue  of 

a  lobe  or  a  large  portion  of  it, 
and  lobular  pneumonia  (bron- 
cho-pneumonia) ,  a  catarrhal 
inflammatory  type  spreading 
from  the  capillary  bronchi  to 
the  air  vesicles  and  often  result- 
ing in  consolidation  of  patches 
of  the  lung  tissue. 

A  number  of  microorganisms 
may  give  rise  to  pneumonia; 
such  for  example  as  B.  mucosus 
capsulatus,  B.  diphtherise,  B. 
pestis,  B.  typhosus,  strepto- 
cocci, and  staphylococci.  For  the  most  part,  however,  these 
organisms  produce  the  lobular  type. 

In  lobar  pneumonia  about  95  per  cent  of  all  the  cases  are  caused 
by  a  lancet-shaped  micrococcus  upon  which  various  names  have 
been  bestowed,  such  as  pneumococcus,  diplococcus  pneumonice, 
micrococcus  lanceolatus,  or  after  its  discoverer,  Frankel's  pneumo- 
coccus. (Fig.  25.) 

The  pneumococcus  was  observed  almost  simultaneously  by 
Pasteur  and  Sternberg  in  1880  in  the  blood  of  rabbits  inoculated 

164 


Fio.  25.  —  Pneumococci. 


PNEUMOCOCCUS  165 

with  human  saliva.  They  did  not,  however,  associate  the  organism 
they  saw  with  lobar  pneumonia.  Later,  in  1886,  Frankel  and 
Weichselbaum  demonstrated  beyond  question  that  the  large 
majority  of  the  cases  of  lobar  pneumonia  are  caused  by  the  pneu- 
mococcus. 

Morphology  and  Staining.  —  Ordinarily  the  organism  appears 
as  a  small,  slightly  oval  coccus,  one  side  of  which  is  somewhat 
pointed.  The  pneumococci  may  occur  singly  or  in  chains,  and 
for  this  reason  they  are  classed  by  certain  authorities  with  the 
streptococci;  usually  they  appear  in  pairs  with  the  broad  ends 
of  the  oval  in  juxtaposition  and  the  pointed  ends  turned  outward. 
When  observed  in  fresh  sputum  or  blood  smears  they  are  sur- 
rounded by  a  well-defined  capsule.  Cultivated  on  artificial 
media  the  capsule  is  rarely  seen  unless  serum  or  blood  has  been 
incorporated  in  the  medium.  The  organisms  are  non-motile, 
possess  no  flagella,  and  do  not  form  spores.  They  stain  readily 
with  the  ordinary  dyes  and  are  Gram  positive.  The  capsule 
may  be  easily  demonstrated  in  blood  or  sputum  preparations  by 
the  method  already  described. 

Cultivation.  —  The  temperature  range  of  the  pneumococcus  is 
rather  limited,  growth  taking  place  as  a  rule  only  between  22°  C. 
and  42°  C.  It  grows  equally  with  or  without  oxygen.  When 
freshly  isolated,  growth  is  very  feeble  unless  blood  or  serum  is 
added  to  the  medium;  on  agar  or  gelatin  the  colonies  appear 
similar  to  those  of  the  pyogenic  cocci  except  that  they  are  more 
delicate  in  appearance.  The  same  may  be  said  of  stab  cultures 
in  gelatin.  Along  the  line  of  inoculation  a  row  of  minute  points 
appear  which  remain  of  a  small  size ;  there  is  no  liquefaction  of 
the  medium.  In  broth  a  slight  turbidity  is  produced  which 
settles  to  the  bottom  of  the  tube  as  a  fine  deposit.  Milk  is  quickly 
acidified  and  often  but  not  always  coagulated ;  growth  on  potato 
is  seldom  visible. 

The  pneumococcus  is  non-hemolytic ;  its  colonies  on  blood 
agar  are  of  a  greenish  color.  Glucose,  saccharose,  lactose,  and 
inulin  are  fermented ;  the  ability  to  ferment  the  latter  sugar  and 
the  fact  that  the  organisms  are  dissolved  in  bile  are  two  important 


166  BACTERIOLOGY   FOR  NURSES 

characteristics  which  aid  in  differentiating  them  from  the  strepto- 
cocci. 

The  pneumococci  may  be  isolated  from  mixed  cultures  or  sputum 
by  smearing  the  material  over  blood  agar  plates.  After  twenty-four 
hours  the  fine  colonies  are  easily  distinguished  from  all  others 
except  the  streptococci;  from  the  latter  they  differ  only  in  that 
they  are  transparent,  a  little  more  delicate  in  appearance,  and 
have  a  smoother  edge. 

By  another  method  a  small  mass  of  sputum  is  washed  free  of 
extraneous  organisms  by  gently  rinsing  it  in  salt  solution  and 
then  injecting  it  into  the  peritoneal  cavity  of  a  white  mouse.  If 
virulent  pneumococci  are  present,  death  will  occur  in  from  twenty- 
four  to  forty-eight  hours  and  the  organisms  will  be  found  in  pure 
culture  in  the  heart's  blood  and  in  the  peritoneal  exudate. 

Resistance.  —  The  pneumococcus  is  decidedly  frail.  Apart  from 
the  body  or  body  discharges  growth  soon  ceases.  On  artificial 
culture  media  it  must  be  transplanted  every  three  or  four  days 
in  order  to  keep  it  alive,  and  even  then  occasional  passage  through 
a  mouse  or  rabbit  is  sometimes  necessary  to  maintain  its  virulence. 
In  dried  sputum  it  may  live  for  several  months  and  retain  its 
pathogenic  power.  Low  temperatures  slightly  above  zero  are 
quite  favorable  to  the  preservation  of  its  vitality.  In  direct 
sunlight  it  dies  within  an  hour.  It  is  quickly  killed  by  a  moderate 
degree  of  heat;  ten  minutes'  exposure  to  52°  C.  is  sufficient. 
Mercuric  chloride  1  to  1000  will  destroy  it  in  five  minutes  and  car- 
bolic acid  1  to  100  in  from  five  to  ten  minutes. 

Pathogenesis.  —  The  pneumococcus  is  frequently  found  in  the 
saliva  of  healthy  individuals.  The  New  York  Commission  reported 
its  presence  in  45  per  cent  of  a  number  of  persons  examined.  Thus 
many  apparently  normal  individuals  are  pneumococcus  "  carriers." 
It  has  been  shown,  however,  that  the  majority  of  such  cases  are 
only  "  temporary  carriers."  The  organism  leaves  the  body  mainly 
in  the  discharges  from  the  mouth  and  nose  and  enters  the  system 
through  the  same  channels. 

Most  strains  are  pathogenic  for  a  number  of  animals.  A  small 
amount  of  sputum  containing  virulent  pneumococci  will  cause 


PNEUMOCOCCUS  167 

the  death  of  a  mouse  or  rabbit  within  twenty-four  to  forty-eight 
hours  from  septicemia.  After  death  the  blood  will  be  found  to 
contain  enormous  numbers  of  organisms. 

Little  is  known  of  the  toxic  substances  produced  by  the  pneumo- 
coccus;  it  is  thought  that  they  are  of  the  nature  of  endotoxins 
and  are  closely  bound  to  the  cell  substance. 

In  characteristic  pneumonia  in  man  the  organisms  are  found 
in  the  bronchioles  and  alveoli  of  the  infected  lung  and  in  the 
lymphatic  channels  and  blood  capillaries;  from  the  capillaries 
they  find  their  way  into  the  general  blood  current.  So  abundant 
are  they  in  a  certain  percentage  of  cases  that  they  may  be  found 
in  cultures  made  from  5  to  10  c.c.  of  blood. 

In  all  cases  of  lobar  pneumonia  and  in  many  cases  of  broncho- 
pneumonia  pleurisy  occurs,  caused  by  the  same  organism  that 
gave  rise  to  the  pneumonia ;  recovery  from  pleurisy  due  to  pneu- 
mococci  is  generally  more  speedy  than  that  caused  by  streptococci 
or  staphylococci. 

Other  infections  frequently  complicating  pneumonia  are  those 
of  the  pericardium,  endocardium,  meninges,  and  middle  ear. 
They  are  probably  explained  by  the  fact  that  the  infecting  organ- 
isms are  conveyed  by  means  of  the  blood  and  lymph  to  all  parts 
of  the  body. 

Immunity.  —  Following  an  attack  of  pneumonia  immunity 
lasts  only  for  a  short  time.  Two  or  three  attacks  of  pneumonia 
are  not  unusual  for  the  same  individual.  A  serum  of  some  pro- 
tective and  curative  value  has  been  produced  by  successive 
injections  of  gradually  increasing  doses  of  virulent  pneumococci 
into  horses.  In  addition,  such  serum  possesses  specific  agglutinins 
which  not  only  are  an  aid  in  diagnosis  but  by  means  of  which  in- 
vestigators have  been  able  to  divide  the  pneumococci  into  four 
groups  according  to  their  specific  reaction.  Group  I  is  the  cause 
of  the  greatest  number  of  infections.  An  immune  serum  has 
already  been  produced  which  has  met  with  considerable  success 
in  conferring  passive  immunity  in  infections  with  this  type.  Fewer 
cases  are  caused  by  Group  II  and  Group  III,  but  the  mortality 
is  much  higher  than  with  Group  I.  A  serum  has  been  prepared 


168  BACTERIOLOGY  FOR  NURSES 

against  Group  II,  but  it  has  not  had  the  same  success  as  that  pre- 
pared against  Group  I.  Group  IV  is  not  really  a  group  but  an 
assembling  together  of  all  the  remaining  isolated  strains. 

Vaccines  have  been  employed  for  the  production  of  active 
immunity  as  a  prophylactic  measure;  their  curative  value  is 
doubtful  in  a  disease  so  acute  and  relatively  brief. 

Relation  of  Pneumococci  and  Streptococci.  —  A  group  of  cocci 
have  frequently  been  found  in  various  diseased  conditions  such 
as  pneumonia  and  meningitis,  which  besides  possessing  a  volu- 
minous capsule  are  surrounded  by  a  viscous  substance  which 
gives  a  slimy  consistency  to  cultures  and  to  exudates.  So  closely 
do  they  appear  to  be  related  both  to  the  pneumococci  and  to  the 
streptococci  that  it  is  difficult  to  determine  with  which  they  should 
be  placed.  It  has  been  suggested  that  they  be  divided  into  two 
groups:  (1)  pneumococcus  mucosus,  which  resembles  the  true 
pneumococcus  in  that  it  is  non-hemolytic  on  blood  agar,  is  soluble 
in  bile,  gives  rise  to  acid  and  coagulation  in  serum  inulin  medium, 
and  is  very  pathogenic  to  white  mice ;  on  the  other  hand,  it  forms 
much  larger  colonies  than  the  pneumococcus,  and  the  individual 
cocci  tend  to  be  less  pointed.  Recent  investigators  regard  it  as 
Group  III  in  the  pneumococcus  classification.  (2)  Streptococcus 
mucosus  is  usually  non-hemolytic,  is  not  soluble  in  bile,  and  does 
not  ferment  inulin;  the  colonies  are  less  transparent,  the  in- 
dividual organisms  are  round  and  occur  in  chains.  Thus  while 
the  pneumococcus  mucosus  is  practically  a  true  pneumococcus 
the  streptococcus  mucosus  appears  to  form  a  connecting  link 
between  it  and  the  streptococci. 


MENINGOCOCCUS 

Inflammation  of  the  membranes  surrounding  the  brain  and 
spinal  cord  may  be  caused  by  several  different  organisms;  it 
may  occur  as  a  primary  or  secondary  infection.  As  a  secondary 
infection  it  not  infrequently  occurs  during  pneumonia  as  a  result 
of  the  pneumococcus  being  carried  to  the  meninges  by  the  blood 
stream;  sometimes  the  tubercle  bacillus  is  the  invader.  In- 


MENINGOCOCCUS  169 

flammation  of  the  middle  ear  or  frontal  sinuses  may  by  extension 
produce  meningitis.  In  such  cases  the  infecting  organisms  are 
usually  staphylococci  or  streptococci.  Sometimes  meningitis  is 
part  of  a  septicemic  or  pyemic  condition ;  occasionally  it  is  due 
to  a  mixed  infection,  and  not  infrequently  the  pneumococcus  has 
been  found  associated  with  the  tubercle  bacillus  and  also  with 
meningococcus. 

It  has  been  estimated  that  about  70  per  cent  of  all  acute  cases 
of  meningitis  appear  in  the  form  designated  epidemic  cerebrospinal 
meningitis,  due  to  the  organism  usually  termed  the  meningococcus. 
In  1884  Weichselbaum  found  the  organism  in  six  cases  of  menin- 
gitis, two  of  which  were  not  complicated  with  pneumonia.  He 
studied  it  in  pure  culture  and  showed  that  it  possessed  character- 
istics which  clearly  distinguished  it  from  the  pneumococcus. 
Because  of  its  frequent  presence  in  the  interior  of  pus  cells  he 
gave  to  it  its  name  of  diplococcus  intracellularis  meningitidis. 

Morphology  and  Staining.  —  The  organisms  appear  as  small 
cocci  usually  arranged  in  pairs,  the  adjacent  sides  being  somewhat 
flattened  against  each  other.  Occasionally  they  are  seen  in  groups 
of  four  or  in  small  masses.  They  are  non-motile,  non-spore- 
bearing,  and  form  no  visible  capsule.  They  stain  with  all  the 
ordinary  dyes  and  are  Gram  negative. 

Cultivation.  —  The  optimum  temperature  for  the  meningococcus 
is  about  37.5°  C. ;  growth  will  occur  between  25°  C.  and  40°  C. 
They  can  rarely  be  isolated  on  plain  nutrient  agar ;  the  addition 
of  a  body  fluid  is  usually  necessary.  On  glucose  ascitic  agar  the 
colonies  appear  as  small,  grayish  white,  finely  granular  disks.  In 
broth  development  is  slow  and  takes  place  near  the  surface. 
Different  strains  vary  in  their  power  to  ferment  carbohydrates 
and  in  their  ability  to  grow  on  artificial  culture  media.  Cultures 
may  remain  alive  for  several  weeks.  Certain  strains,  however, 
tend  to  die  within  three  or  four  days  and  consequently  require 
transplanting  to  a  fresh  medium  at  very  short  intervals. 

Resistance.  —  The  organism  is  readily  destroyed  by  sunlight 
or  drying  or  by  exposure  to  a  moderate  degree  of  heat  or  cold. 
It  is  killed  in  from  one  to  five  minutes  by  1  to  1000  solution  of 


170  BACTERIOLOGY  FOR  NURSES 

mercuric  chloride  or  1  to  100  solution  of  carbolic  acid.  Its  low 
resistance  to  influences  outside  of  the  body,  together  with  the 
fact  that  it  has  not  been  demonstrated  in  the  air  or  dust,  is  an 
evidence  of  its  parasitic  nature. 

Pathogenesis.  —  The  organisms  vary  in  their  pathogenicity 
for  animals.  Certain  strains  injected  into  the  peritoneal  cavity 
of  a  guinea  pig  will  produce  a  septicemia.  Killed  cultures  may 
also  have  a  fatal  effect,  in  which  case  death  is  probably  due  to  a 
bacterial  poison  of  the  nature  of  endotoxin,  liberated  by  the  dis- 
integration of  the  organism. 

In  human  beings  the  course  of  the  disease  is  very  rapid;  the 
lesion  is  of  a  suppurative  nature  involving  the  meninges,  the 
base  of  the  brain,  and  the  surface  of  the  spinal  cord.  During  life 
the  surest  method  of  diagnosis  is  the  detection  of  the  specific 
microorganism  in  the  spinal  fluid  withdrawn  by  means  of  a  lumbar 
puncture.  In  cases  of  meningitis  due  to  the  meningococcus,  the 
fluid  appears  somewhat  cloudy,  contains  a  high  percentage  of 
polynuclear  leukocytes,  and  the  characteristic  Gram  negative 
diplococci  free  or  engulfed  within  the  leukocytes.  The  mortality 
without  serum  treatment  is  about  70  per  cent. 

The  organisms  in  all  probability  enter  the  body  by  way  of  the 
nasopharynx,  passing  out  of  the  nose  and  its  adjoining  cavities 
along  the  path  of  the  lymphatics  toward  the  base  of  the  skull. 
They  are  present  in  great  numbers  in  the  nasal  cavity  during  the 
first  twelve  days  of  the  disease,  after  which  they  disappear. 

Epidemics  usually  occur  in  the  winter  and  spring  months  and 
commence  in  localities  where  overcrowding  is  most  likely.  Be- 
cause of  the  low  vitality  of  the  organism  outside  of  the  body 
"  carriers  "  may  be  largely  responsible  for  the  majority  of  out- 
breaks, since  the  disease  is  undoubtedly  transmitted  from  person 
to  person.  During  an  epidemic  not  all  the  persons  who  harbor 
the  organism  develop  the  disease ;  the  carriers  have  been  shown 
to  outnumber  the  actual  cases  by  ten  to  one.  Apart  from  epi- 
demics the  meningococcus  is  rarely  found  on  the  membranes  of 
healthy  persons,  but  evidently  there  are  those  who  carry  it  always 
and  thus  perpetuate  the  disease. 


MENINGOCOCCUS  171 

Agglutinins.  —  An  agglutination  reaction  towards  the  specific 
strain  of  meningococci  causing  the  disease  is  often  given  if  death 
does  not  occur  within  the  first  few  days.  A  positive  reaction  may 
appear  by  the  fourth  day  in  a  dilution  of  1  to  50 ;  at  a  later  stage 
it  may  even  occur  in  as  great  a  dilution  as  1  to  400.  A  certain 
number  of  strains,  however,  do  not  give  the  reaction,  and  for  this 
reason  the  test  is  unreliable  and  practically  never  used  for  diagnosis. 

An  antiserum  has  been  prepared  by  injecting  horses  with  a 
mixture  of  several  strains  of  meningococci.  A  serum  prepared 
by  Flexner  and  Jobling  has  been  extensively  used  both  in  Amer- 
ica and  Europe  and  has  given  very  satisfactory  results;  the  use 
of  such  an  antimeningococcic  serum  has  reduced  the  mortality 
from  70  to  30  per  cent  and  has  greatly  diminished  the  tendency 
to  chronic  lesions  in  those  who  survive. 

To  administer  the  antiserum  a  lumbar  puncture  is  made  in 
about  the  third  or  fourth  lumbar  space,  the  cerebrospinal  fluid 
is  allowed  to  flow  until  only  about  three  or  four  drops  come  per 
minute,  and  then  the  serum,  which  has  been  previously  warmed 
to  body  temperature,  is  allowed  to  flow  in  by  gravity.  The 
average  dose  for  a  child  is  from  2  to  20  c.c.  and  for  an  adult  from 
20  to  40  c.c.  More  depends  on  the  amount  of  fluid  withdrawn 
than  the  age  of  the  patient.  In  severe  cases  the  serum  is  injected 
every  twelve  hours  until  there  is  an  improvement;  in  milder 
cases  it  is  repeated  each  day  for  the  first  four  days.  Usually 
from  four  to  six  injections  are  necessary  although  as  many  as 
fifteen  have  been  employed. 

Vaccines.  — As  a  prophylactic  measure  three  injections  at 
weekly  intervals  of  250  millions,  500  millions,  and  1  billion  re- 
spectively have  been  advocated.  In  cases  where  lumbar  puncture 
and  serum  treatment  has  had  little  effect  an  autogenous  vaccine 
is  sometimes  employed  therapeutically. 

THE   GONOCOCCUS 

Gonorrhea  is  one  of  the  most  widely  disseminated  of  all 
the  infectious  diseases.  When  it  was  first  recognized  is  not 


172 


BACTERIOLOGY  FOR  NURSES 


known ;  mention  is  made  of  it,  however,  in  the  earliest  medical 
records. 

Neisser  in  1879  described  a  coccus  constantly  present  in  the 
pus  of  gonorrheal  infections,  to  which  he  gave  the  name  of  gono- 
coccus. 

In  1885  Bumm  succeeded  in  isolating  it  and  cultivating  it  upon 
coagulated  human  blood  serum.  Later  he  proved  its  specificity 
beyond  doubt  by  inoculating  it  into  men  and  producing  the 
characteristic  disease. 

Morphology  and  Staining.  —  The  organism  usually  occurs  in 
the  form  of  a  diplococcus  closely  resembling  the  meningococcus. 

The  adjacent  sides  of  the  two 
cocci  appear  to  be  slightly 
concave,  so  that  in  stained 
preparations  they  have  some- 
what the  appearance  of  two 
coffee  beans  placed  side  by 
side.  In  the  early  stages  of 
the  disease  the  organisms  ap- 
pear free  or  lying  on  the  sur- 
face of  desquamated  epithelial 
cells.  When  the  secretion  be- 
comes purulent  they  may  be 
seen  within  the  protoplasm  of 
the  leukocytes  in  such  num- 
bers that  the  latter  may  appear  to  be  filled  with  them.  As  the 
disease  becomes  more  chronic  the  phagocytes  appear  to  become 
less  active  and  fewer  organisms  are  found  engulfed  within  them. 
(Fig.  26.) 

The  gonococcus  is  not  motile,  does  not  produce  spores,  is  easily 
stained  with  any  of  the  basic  dyes,  and  is  Gram  negative. 

Cultivation.  —  Growth  takes  place  best  at  body  temperature ; 
below  25°  C.  and  above  40°  C.  no  development  occurs.  It  is 
advisable  to  inoculate  media  as  soon  as  possible  after  obtaining 
material  from  the  body  and  to  place  the  tubes  in  the  incubator 
at  once. 


FIG.  26.  —  Gonococci  within  and  near  to 
Leukocytes. 


GONOCOCCUS  173 

It  is  seldom  a  strain  of  the  gonococcus  will  grow  on  ordinary 
nutrient  agar;  as  a  rule  it  requires  the  addition  of  blood  serum 
or  other  body  fluid.  Colonies  appear  within  twenty-four  to 
forty-eight  hours  as  delicate,  finely  granular  disks  with  scalloped 
margins ;  in  color  they  are  grayish  white  with  a  tinge  of  yellow. 
When  freshly  isolated  the  organisms  die  in  from  two  to  three 
days  unless  transplanted ;  older  cultures  may  live  three  weeks  and 
if  kept  in  the  ice  box  even  longer. 

Comparison  with  Meningococcus.  —  Morphologically  and  cul- 
turally the  two  organisms  resemble  each  other  very  closely.  The 
following  points  are  of  importance  in  differentiating  them.  The 
meningococcus  grows  on  culture  media  more  readily  than  the 
gonococcus.  After  the  first  subculture  it  will  frequently  grow  on 
nutrient  agar,  whereas  the  gonococcus  will  rarely  grow  on  ordinary 
agar.  The  colonies  of  the  latter  are  less  opaque  and  have  a  more 
irregular  margin  than  those  of  the  meningococcus.  The  meningo- 
coccus grows  well  in  broth  with  a  neutral  reaction,  producing  a 
general  turbidity;  whereas  the  gonococcus  does  not  grow,  and 
even  if  serum  is  added  growth  is  very  scanty  and  falls  to  the 
bottom  as  a  deposit,  leaving  the  medium  clear.  Of  the  sugars 
usually  employed  the  gonococcus  ferments  glucose  only,  the 
meningococcus  ferments  maltose  also.  As  a  rule  the  part  of  the 
body  from  which  the  organisms  are  obtained  gives  sufficient 
information. 

Resistance.  —  The  gonococcus  is  very  feebly  resistant  to 
harmful  influences.  It  is  quickly  destroyed  by  drying  when  in 
thin  layers  of  pus ;  in  comparatively  thick  layers  smeared  on  linen 
it  has  been  found  alive  after  several  days.  The  organism  has 
never  been  found  apart  from  the  human  body  or  material  ob- 
tained from  it. 

Pathogenesis.  —  All  attempts  to  reproduce  the  disease  in  lower 
animals  has  so  far  failed.  Intraperitoneal  injections  of  living 
cultures  into  white  mice  produce  peritonitis,  but  the  organisms 
appear  to  be  unable  to  multiply  and  soon  disappear.  Injected 
into  the  joints  of  rabbits  and  dogs,  they  cause  an  acute  inflamma- 
tion which  soon  subsides  and  the  gonococci  can  no  longer  be 


174  BACTERIOLOGY  FOR  NURSES 

found.  A  similar  result  is  obtained  when  dead  cultures  are  used. 
Thus  it  is  evident  that  while  large  numbers  of  the  organisms  can 
produce  a  certain  amount  of  inflammation  they  are  unable  to 
multiply  and  spread  in  animal  tissues. 

In  human  beings  infection  is  usually  a  result  of  sexual  inter- 
course. Gonorrhea  in  men  frequently  results  in  prostatitis  and 
epididymitis ;  in  women  vaginitis  and  endocervicitis,  or  spreading 
by  direct  continuity  of  tissue,  the  Fallopian  tubes,  the  ovaries, 
or  even  the  peritoneum  may  become  involved.  Sterility  may 
result  as  a  consequence  of  a  salpingitis  which  obstructs  the 
Fallopian  tubes,  or  in  man  epididymitis  may  have  a  similar 
result. 

The  organisms  penetrate  the  mucous  membranes  and  passing 
between  the  epithelial  cells  cause  an  inflammatory  reaction  in  the 
tissues  below.  Secretion  increases  and  numbers  of  leukocytes 
gather  around  the  infected  area.  The  infection  may  remain  more 
or  less  localized  or  the  organisms  may  be  carried  through  the 
blood  and  lymph  to  more  distant  parts  of  the  body.  One  of  the 
most  serious  and  disabling  complications  is  gonorrheal  arthritis; 
neuralgic  affections,  muscle  atrophies,  and  neuritis  often  accom- 
pany or  follow  a  gonorrheal  infection. 

In  gonorrheal  conjunctivitis  microscopic  examination  of  the 
secretion  generally  reveals  the  identity  of  the  invading  organism. 
As  the  condition  becomes  chronic  the  gonococci  are  less  numerous 
and  a  greater  proportion  of  other  organisms  appear.  Of  all  cases 
of  ophthalmia  neonatorum  about  two  thirds  are  caused  by  the 
gonococcus. 

Immunity.  —  After  recovery  from  an  infection  immunity  seems 
to  be  very  slight  if  indeed  it  exists.  Gonococci  may  persist  in 
the  genito-urinary  secretions  for  years  after  apparent  recovery 
has  taken  place  and  may  at  any  time  cause  an  acute  gonorrhea 
in  another  person  or  even  in  the  individual  carrying  them.  There 
is  no  limit  to  the  time  an  individual  may  remain  infected  or  may 
infect  others. 

A  slightly  bactericidal  serum  has  been  produced  by  animal 
inoculations.  A  moderate  degree  of  passive  immunity  is  said  to 


GONOCOCCUS  175 

result  from  its  use  in  cases  of  arthritis;  in  acute  gonorrhea  it  has 
no  effect  whatever. 

Vaccines.  —  Vaccines  have  been  employed  with  good  results 
in  joint  inflammations  and  chronic  lesions  of  the  urethra  and 
bladder.  They  are  more  effective  when  prepared  with  the  organ- 
isms infecting  the  patient  to  be  treated.  Experiments  have  shown 
that  at :  least  ten  different  strains  of  gonococci  exist.  Hence  a 
polyvalent  vaccine  in  which  each  type  is  represented  is  advisable 
if  an  autogenous  vaccine  is  not  employed.  The  dose  is  from  25 
to  500  millions,  increasing  to  1  billion  every  three  to  seven 
days. 

Other  Pathogenic  Micrococci  Resembling  the  Meningococcus 
and  Gonococcus.  Micrococcus  catarrhalis.  The  organisms  usually 
occur  in  pairs,  resemble  the  meningococcus  in  form,  and  are  nega- 
tive to  Gram's  stain.  On  nutrient  agar  they  will  develop  between 
20°  and  40°  C.  and  appear  as  small  yellowish  white  colonies ;  on 
serum  agar  their  growth  is  much  more  luxuriant.  They  are  often 
found  on  the  mucous  membrane  of  the  respiratory  tract  and  not 
infrequently  excite  a  catarrhal  inflammation. 

Micrococcus  Melitensis.  —  In  1887  Bruce  discovered  the 
organism  in  a  case  of  Malta  fever.  It  was  formerly  thought 
that  the  disease  was  confined  to  the  shores  of  the  Mediterranean 
and  its  islands.  Cases  have  occurred,  however,  in  India,  China, 
South  Africa,  and  some  parts  of  North  and  South  America.  The 
organism  is  a  slightly  oval  coccus  occurring  singly  or  in  pairs, 
non-motile  and  negative  to  Gram's  stain.  Its  growth  is  slow. 
Colonies  on  agar  are  not  visible  until  the  third  day,  when  they 
appear  as  small  round  spots,  somewhat  transparent  with  bluish 
white  margin  and  yellowish  tinted  center.  The  organism  is 
destroyed  by  a  temperature  of  60°  C.  in  twenty  minutes,  and  by 
carbolic  acid  1  to  100  in  fifteen  minutes.  It  shows  rather  a  marked 
resistance  to  drying. 

The  milk  of  goats  is  considered  the  chief  source  of  infection, 
although  the  disease  may  be  conveyed  by  other  means.  A  case 
is  reported  as  contracted  by  the  use  of  a  clinical  thermometer; 
a  fatal  case  occurred  as  a  result  of  laboratory  infection. 


176  BACTERIOLOGY  FOR  NURSES 

In  man  the  disease  appears  as  an  intermittent  fever,  often 
accompanied  by  pains  of  a  rheumatic  or  neuralgic  character. 
The  fever  usually  lasts  from  one  to  three  weeks  and  may  recur 
from  time  to  time  during  a  period  of  several  months.  The  or- 
ganisms appear  in  the  blood  at  the  height  of  the  fever  and  are 
present  in  various  organs  and  in  the  urine  from  the  second  day 
to  the  end  of  the  disease.  Autopsies  reveal  degeneration  both 
of  the  liver  and  spleen. 

As  an  aid  to  diagnosis,  blood  cultures  are  usually  made  during 
the  period  the  fever  is  highest.  A  typical  characteristic  of  Malta 
fever  is  the  appearance  of  agglutinins  in  the  serum,  which  give  a 
marked  reaction  in  high  dilutions.  The  serum  of  a  patient  may 
agglutinate  the  micrococcus  melitensis  in  a  dilution  as  high  as 
1  to  1000.  Animals  injected  with  the  organism  will  produce  a 
serum  which  will  react  in  dilutions  as  high  as  1  to  100,000.  By 
this  means  suspected  cultures  can  be  readily  identified. 


CHAPTER  XVII 
THE   DIPHTHERIA  BACILLUS 

DIPHTHERIA  under  various  names  has  been  described  almost 
since  the  earliest  days  of  history.  In  1821  Bretonneau  of  Tours 
published  a  very  comprehensive  essay  on  the  subject  and  gave 
to  the  disease  its  present  name.  Little  further  information  was 
gained  until  about  1840.  Observers  began  to  notice  the  presence 
of  microorganisms  in  the  pseudomembranes  and  suggested  they 
might  be  the  causal  agents.  In  1883  Klebs  described  a  bacillus 
of  rather  peculiar  appearance  which  could  be  almost  invariably 
demonstrated  in  the  false  membrane  of  the  throats  of  those  dying 
of  true  diphtheria.  A  year  later  the  organism  was  isolated  and 
cultivated  in  pure  culture  by  Loeffler,  who  described  its  character 
and  its  pathogenic  effects  on  animals.  Loeffler  was  able  by  inocu- 
lation with  the  bacillus  to  produce  the  false  membrane  on  damaged 
mucous  surfaces.  But  he  hesitated  to  conclude  definitely  that  the 
organism  was  the  direct  cause  of  the  disease,  because  he  was  not 
able  to  find  it  in  every  case  thought  to  be  diphtheria,  and  also  he 
had  found  it  in  the  throat  of  a  normal,  healthy  child.  Such  condi- 
tions are  more  clearly  understood  now.  Similar  clinical  symptoms 
are  not  necessarily  produced  by  the  same  agent,  and  the  knowledge 
recently  gained  that  healthy  persons  may  become  carriers  explains 
the  occasional  appearance  of  the  organism  in  normal  throats. 
Additional  confirmatory  evidence  was  given  when  Roux  and 
Yersin  in  1889-1890  showed  that  the  most  important  features  of 
the  disease  could  be  produced  by  means  of  the  toxins  separated 
from  the  organisms.  By  clinical  and  bacterial  observations  the 
relationship  of  the  Klebs-Loeffler  bacillus  to  the  disease  is  now  so 
definitely  established  that  it  has  become  a  necessity  to  find  the 
N  177 


178 


BACTERIOLOGY   FOR  NURSES 


organism  in  the  lesion  before  a  diagnosis  of  diphtheria  can  with 
certainty  be  made. 

Morphology  and  Staining.  —  The  diphtheria  bacillus  is  a  slender, 
straight  or  slightly  curved  rod  ranging  from  1  /*  to  6  /*  in  length. 
The  different  strains  vary  considerably  in  form  and  even  the  same 
strain  may  assume  a  somewhat  different  shape  under  changed 
conditions.  Freshly  isolated  organisms  often  possess  granules 
which  give  them  a  beaded  appearance.  Others  that  have  been 
grown  on  culture  media  may  develop  swollen  ends  that  give  to 
them  the  appearance  of  an  Indian  club  ;  others  again  are  thicker  in 

the  center  and  taper  at  one  or 
both  ends.  When  thickened 
at  one  end  only  they  appear 
somewhat  like  a  wedge. 
Stained  with  Loeffler's  meth- 
ylene  blue  the  bacilli  may 
appear  uniformly  colored  or 
they  may  present  a  barred 
or  striated  appearance.  The 
round  bodies  in  the  granu- 
lated forms  (metachromatic 
granules)  stain  much  more 
intensely  than  the  rest  of  the 
organism.  This  peculiarity 
of  form  and  staining  appears  to  have  a  certain  relation  to  the 
period  of  growth.  A  twelve-hour  culture  is  most  likely  to  show 
granular  forms.  A  twenty-four-hour  growth  will  show  more  club 
forms  than  at  twelve  hours.  Older  cultures  still  stain  very 
faintly.  Thus  one  may  often  see  in  a  stained  preparation  the  dif- 
ferent forms  side  by  side.  The  round  or  oval  bodies  which  are 
so  intensely  colored  with  methylene  blue  appear  even  more  dis- 
tinct when  Neisser's  stain  is  used.  Colored  by  the  latter  method 
the  granules  are  almost  black,  while  the  remainder  of  the  bacillary 
substance  is  of  a  yellowish  brown.  Serum  cultures  of  about  twelve 
hours'  growth  should  be  employed  for  this  method.  It  was  orig- 
inally thought  that  the  presence  of  granules  in  the  bacilli  indicated 


FIG.  27.  —  Diphtheria  Bacilli. 


THE  DIPHTHERIA  BACILLUS  179 

a  degree  of  virulence,  and  that  by  the  use  of  Neisser's  stain  the 
virulent  forms  of  the  diphtheria  bacilli  could  thus  be  distinguished 
from  the  non- virulent  without  the  delay  of  inoculating  animals. 
Experiments  have  shown,  however,  that  the  variation  in  form  and 
staining  properties  has  no  relation  to  pathogenicity  and  that  by 
no  means  at  present  known  can  the  virulent  strains  be  distin- 
guished from  the  non-virulent  except  by  animal  inoculations. 
The  bacilli  are  non-motile  and  do  not  form  spores.  (Fig.  27.) 

Cultivation.  —  Growth  takes  place  best  at  body  temperature. 
Development  will  occur  at  a  temperature  as  low  as  20°  C. ;  below 
that,  however,  it  usually  ceases.  The  organism  is  aerobic  and 
facultative  anaerobic.  When  freshly  isolated  it  grows  much  more 
readily  on  media  containing  serum.  The  mixture  of  Loeffler 
has  been  found  to  be  one  of  the  best  for  making  cultures  direct 
from  suspected  throats.  At  the  end  of  about  twelve  hours  colonies 
of  the  diphtheria  bacilli  appear  as  pearl-gray  or  occasionally  yel- 
lowish gray,  slightly  raised  points  a  little  larger  than  the  colonies 
of  streptococci  and  a  little  smaller  than  those  of  the  staphylococci. 

On  gelatin  at  22°  C.  a  stab  culture  shows  a  beaded  appearance 
along  the  line  of  inoculation,  while  at  the  surf  ace 'growth  forms  a 
small  disk.  No  liquefaction  occurs.  Milk  is  an  excellent  medium. 
Growth  is  rapid  and  luxuriant ;  the  lactose  is  not  fermented  nor  is 
the  casein  coagulated.  In  broth  a  cloudiness  is  first  produced 
which  soon  settles  to  the  bottom  and  along  the  sides  of  the  tube  as 
a  fine,  powdery  deposit.  If  the  broth  is  inoculated  on  the  surface 
and  the  tube  is  allowed  to  remain  undisturbed  growth  is  apt  to  occur 
as  a  fine  but  distinct  scum  upon  slightly  alkaline  nutrient  agar  to 
which  has  been  added  1  per  cent  dextrose.  Good  growth  will 
result  after  one  or  two  generations  have  been  cultivated  on  serum 
media.  The  appearance  of  the  colonies  on  agar  is  peculiarly 
characteristic  and  for  this  reason  it  is  of  value  for  the  isolation  of 
the  organism.  Surface  colonies  appear  to  have  a  dark,  coarsely 
granular,  piled-up  center  with  a  thin  irregular  border  which  some- 
times appears  jagged  or  torn. 

Isolation.  —  Petri  plates  are  first  prepared  by  pouring  into  them 
nutrient  glucose  agar  and  allowing  it  to  solidify.  If  the  mixed 


180  BACTERIOLOGY  FOR  NURSES 

culture  has  been  grown  on  ascitic  broth  a  small  portion  of  the 
pellicle  is  removed  on  a  platinum  loop  and  lightly  streaked  over 
several  of  the  prepared  plates;  if  instead  of  broth  the  mixed 
organisms  are  removed  from  serum  medium  the  portion  showing 
colonies  which  most  resemble  those  of  the  diphtheria  is  chosen. 
The  plates  are  incubated  for  about  sixteen  hours  at  37°  C.,  after 
which  the  most  characteristic  colonies  are  "  fished  "  and  trans- 
ferred either  to  Loeffler's  serum  tubes  or  to  the  ascitic  broth. 

Resistance.  —  In  cultures  the  bacilli  will  live  for  a  long  time 
at  room  temperature.  They  may  survive  for  two  months  or  more 
without  transplanting.  They  are  particularly  resistant  to  cold 
at  temperatures  just  below  freezing;  they  will  remain  alive  for 
weeks.  In  a  moist  condition,  whether  in  cultures  or  a  membrane, 
their  resistance  to  heat  is  comparatively  low.  Ten  minutes'  ex- 
posure to  a  temperature  of  60°  C.  is  sufficient  to  destroy  them. 
On  the  other  hand,  in  a  dry  condition  they  possess  a  much  greater 
power  of  endurance.  In  a  membrane  which  is  perfectly  dry  they 
can  resist  a  temperature  of  98°  C.  for  one  hour.  Vigorous  toxic 
diphtheria  bacilli  have  been  found  on  dried  membrane  four  months 
after  its  removal  from  a  throat.  On  toys,  pencils,  paper  money, 
etc.,  they  may  live  for  several  weeks.  Their  resistance  to  dis- 
infectants is  much  the  same  as  that  of  other  non-spore-bearing 
bacteria.  They  are  killed  in  a  solution  of  mercuric  chloride 
1  to  1000  in  from  one  to  five  minutes  and  in  carbolic  1  to  100  in 
from  five  to  ten  minutes. 

Pathogenesis.  —  With  the  exception  of  rats  and  mice  most 
of  the  lower  animals  are  susceptible  to  the  toxin  of  the  diphtheria 
bacillus,  yet  it  is  extremely  rare  that  the  disease  appears  in  them. 
In  fact  the  cat  is  the  only  animal  known  to  have  contracted  diph- 
theria from  contact  with  the  disease.  False  membranes  similar 
to  those  produced  by  the  diphtheria  bacillus  in  human  beings  may 
occur  in  animals,  but  only  when  the  membrane  has  been  first 
abraded  and  then  virulent  organisms  either  rubbed  on  to  it  or  in- 
jected into  it. 

Very  small  quantities  of  a  virulent  broth  culture  injected  sub- 
cutaneously  will  produce  symptoms  of  toxemia  in  a  guinea  pig 


THE  DIPHTHERIA   BACILLUS  181 

within  six  to  eight  hours.  If  the  animal  does  not  succumb  to  a 
rapid  intoxication  signs  of  paralysis  appear  in  the  lower  extremi- 
ties, gradually  extending  to  the  entire  body,  and  causing  death  by 
paralysis  of  the  heart  or  respiratory  muscles.  Upon  autopsy  the 
site  of  inoculation  is  found  to  be  congested  and  the  neighboring 
lymph  nodes  swollen;  the  adrenals  are  congested;  an  excess  of 
fluid  appears  in  the  serous  cavities  and  in  the  heart;  voluntary 
muscle  fibers  and  nervous  tissue  show  signs  of  degeneration. 

In  human  infection  the  disease  is  characterized  by  a  pseudo- 
membrane  on  a  mucous  membrane,  or  occasionally  upon  the 
surface  of  a  wound  and  a  general  toxemia.  The  site  of  the  pseudo- 
membrane  is  usually  the  throat,  larynx,  or  nose ;  diphtheritic  in- 
fection of  the  middle  ear  is  not  uncommon ;  infection  of  the  con- 
junctiva sometimes  occurs  as  a  result  of  a  patient's  coughing  or 
sneezing  into  the  eye  of  another  person.  The  local  lesion  is 
the  result  of  bacterial  invasion,  and  consequent  degeneration  of 
the  epithelial  cells  gradually  extends  to  the  underlying  tissues. 
A  profuse  fibrinous  exudate  is  poured  out,  and  soon  spreading  over 
the  surface  a  false  membrane  appears  composed  of  fibrin,  leukocytes, 
dead  tissue  cells,  and  bacteria.  The  pseudomembrane  may  be 
so  thick  and  firmly  adherent  as  to  leave  a  torn  and  bleeding  sur- 
face when  displaced. 

The  most  serious  injuries  caused  by  the  diphtheria  bacillus  are 
the  systematic  lesions  due  to  the  absorption  of  its  poisons.  Diph- 
theria is  primarily  a  toxemia.  As  a  result  fatty  degeneration 
takes  place  in  the  muscle  fibers  of  the  heart,  in  the  myelin  sheath 
of  the  peripheral  nerves,  and  in  the  white  matter  of  the  brain  and 
spinal  cord  and  in  the  kidneys.  These  changes  in  muscle  and 
nerve  explain  the  paralysis  and  cardiac  weakness  so  often  follow- 
ing an  attack  of  diphtheria.  When  death  occurs  as  a  result  of 
the  infection  it  is  usually  due  to  toxemia,  laryngeal  obstruction, 
or  broncho-pneumonia. 

Diphtheria  Toxin.  —  Diphtheria  bacilli  when  growing  in  nutrient 
broth  produce  a  soluble  toxin  which  diffuses  from  their  bodies 
into  the  surrounding  medium.  Loeffler  assumed  the  presence  of 
such  a  poison  but  Roux  and  Yersin  were  the  first  to  obtain  it  apart 


182  BACTERIOLOGY  FOR  NURSES 

from  the  living  bacilli  by  filtration  through  a  porcelain  filter. 
Little  is  known  regarding  its  chemical  nature,  but  it  has  many  of 
the  properties  of  protein  substances.  It  is  completely  destroyed 
by  boiling  for  five  minutes  and  loses  a  great  deal  of  its  strength 
when  heated  to  75°  C.  Its  toxicity  is  lost  when  exposed  for  a 
few  hours  to  direct  sunlight.  On  the  other  hand,  kept  in  cool  and 
dark  storage  it  deteriorates  very  slowly. 

The  symptoms  produced  in  animals  are  practically  the  same 
whether  cultures  of  living  bacilli  or  the  germ-free  toxin  be  injected, 
except  that  when  toxin  only  is  introduced  no  false  membrane  is 
formed.  The  lesions  which  occur  in  the  heart  and  other  organs 
are  identical ;  consequently  there  is  sufficient  proof  that  the  chief 
injury  to  the  body  is  caused  by  the  powerful  poison  secreted 
by  the  living  bacterial  cells  grouped  together  in  enormous  numbers 
in  the  false  membrane  of  the  throat.  The  organisms  pour  out 
their  poison,  which  readily  passes  into  the  underlying  tissues  and 
diffuses  through  the  body,  injuring  particularly  those  cells  for 
which  it  has  a  special  affinity. 

There  is  a  wide  variation  in  the  ability  of  diphtheria  bacilli  to 
produce  toxin.  The  great  majority  of  organisms  isolated  from 
throat  exudates  or  pseudomembranes  which  possess  the  charac- 
teristics of  the  diphtheria  bacillus  are  found  to  be  strongly  toxic. 
There  are,  however,  grades  of  toxicity  until  finally  we  reach  a  small 
group  sometimes  found  in  slightly  inflamed  or  normal  throats 
which  are  morphologically  and  culturally  identical  with  the 
Klebs-Loeffler  bacillus  yet  do  not  produce  in  culture  media  or  test 
animals  the  diphtheria  toxin.  From  a  public  health  standpoint 
such  organisms  are  harmless,  since  it  has  not  been  proven  that  a 
non-toxin  producer  ever  develops  the  power.  It  may  be  that 
the  ancestors  of  these  organisms  were  true  diphtheria  bacilli  and 
that  succeeding  generations  have  by  attenuation  lost  the  power  of 
producing  toxin.  That,  however,  is  only  a  supposition.  Certain 
investigators  claim  that  a  true  diphtheria  bacillus  never  completely 
loses  its  ability  to  produce  toxin,  however  attenuated,  and  that 
related  bacilli  which  do  not  possess  the  power  never  gain  it.  The 
passage  of  diphtheria  bacilli  through  the  body  of  a  susceptible 
animal  has  little  effect  on  their  toxin  production. 


THE  DIPHTHERIA  BACILLUS  183 

The  severity  of  the  disease  produced  by  the  diphtheria  bacillus 
cannot  be  regarded  as  an  index  of  the  virulence  of  that  particular 
strain.  Association  with  other  organisms  and  the  presence  of 
varying  amounts  of  antitoxin  in  the  blood  of  the  patient  may  mask 
the  real  power  of  the  invader.  Descendants  of  the  same  organism 
may  give  rise  to  mild  symptoms  in  one  person  and  to  a  fatal  infec- 
tion in  another. 

Persistence  of  Diphtheria  Bacilli  in  the  Throat.  —  The  length 
of  time  the  bacilli  continue  to  live  in  the  throat  after  apparent 
recovery  varies  greatly.  "  Diphtheria  bacilli  disappear  in  about 
50  per  cent  of  cases  by  the  time  the  local  membrane  has  dis- 
appeared. They  persist  in  about  5  per  cent  of  persons  at  the  end 
of  two  months,  about  2  per  cent  at  the  end  of  three  months,  and 
approximately  1  per  cent  continue  as  chronic  bacillus  carriers." 
(Rosenau.) 

Immunity.  —  The  fact  that  fully  toxic  bacilli  have  been  fre- 
quently found  in  the  throats  of  healthy  persons  who  have  been 
brought  in  contact  with  diphtheria  patients,  yet  who  have  not  con- 
tracted the  disease,  demonstrates  that  diphtheria,  like  other  infec- 
tious diseases,  requires  not  only  the  presence  of  the  specific  organ- 
ism but  also  a  susceptibility  on  the  part  of  the  individual.  It  is 
estimated  that  about  70  per  cent  of  all  persons  are  protected  from 
infection  because  of  an  antitoxin  present  in  their  blood.  Condi- 
tions therefore  which  impair  vitality  and  diminish  the  production 
of  specific  antibodies  increase  susceptibility. 

Immunity  following  an  attack  of  diphtheria  usually  lasts  for 
several  months  or  even  years.  Occasionally,  however,  it  is  of 
much  shorter  duration.  Infants  and  adults  possess  relatively 
more  immunity  than  young  children  between  the  ages  of  two  and 
ten  years.  It  is  known  that  young  animals  born  of  immunized 
mothers  inherit  a  certain  degree  of  resistance.  This  may  explain 
the  relative  insusceptibility  of  children  during  the  first  months  of 
life.  Passive  immunity  is  only  of  short  duration;  the  antitoxin 
injected  usually  disappears  from  the  blood  in  less  than  three  weeks. 
The  nature  of  antitoxin  and  its  prophylactic  and  therapeutic  use 
is  discussed  in  Chapter  II. 


184  BACTERIOLOGY  FOR  NURSES 

Mixed  Infections.  —  The  diphtheria  bacillus  is  not  the  only 
organism  usually  found  in  the  false  membrane.  Associated  with 
it  are  frequently  found  the  pyogenic  cocci.  The  streptococcus 
is  an  especially  useful  ally  in  disintegrating  the  surface  cells  of 
the  mucous  membrane,  and  making  possible  the  penetration  of 
the  diphtheria  bacillus  into  the  deeper  tissues,  thus  facilitating  the 
absorption  of  its  toxin.  Certain  suppurative  conditions  of  the 
throat  are  unquestionably  due  to  the  pyogenic  cocci.  In  most 
cases  of  fatal  broncho-pneumonia  following  diphtheria  streptococci 
or  pneumococci  or  both  are  usually  the  inciting  organisms  and  as 
diphtheria  antitoxin  has  absolutely  no  effect  on  them  they  fre- 
quently are  the  cause  of  death.  The  presence  of  certain  other 
bacteria  may  often  be  detected  by  a  difference  in  the  exudate; 
for  example :  B.  fusiformis  gives  rise  to  an  offensive  odor,  B. 
pyocyaneus  to  a  bluish  green  color. 

Bacteriological  Diagnosis.  —  A  pseudomembrane  in  the  nose 
or  throat  is  usually  but  not  always  the  result  of  an  infection  by 
the  Loeffler  bacillus.  Many  other  organisms  frequently  present 
in  the  throat  secretions,  such  for  example  as  streptococci  and 
pneumococci,  can  under  certain  conditions  produce  a  local  lesion 
very  similar  to  that  of  a  mild  case  of  diphtheria.  Vincent's  angina 
and  the  pseudomembrane  in  scarlet  fever  somewhat  resemble 
that  produced  by  the  diphtheria  bacillus.  Generally,  however, 
the  deposit  in  the  first-named  diseases  appears  rather  as  an  exudate 
than  a  membrane. 

Nearly  all  membranous  affections  of  the  nose  are  diphtheritic, 
as  are  also  thick,  grayish  membranes  spreading  over  a  large  portion 
of  the  tonsils  and  the  soft  palate.  Seen  on  the  tonsils  alone  the 
presence  of  the  diphtheria  bacillus  is  less  sure. 

In  uncertain  cases  bacterial  examination  is  of  the  greatest  value 
since  the  disease  has  such  a  rapid  onset  and  the  early  adminis- 
tration of  antitoxin  is  necessary  both  for  the  suspected  case  and 
those  who  may  come  in  contact  with  it. 

The  examination  of  cultures  made  from  suspected  throats  is 
usually  a  routine  procedure  in  municipal  laboratories.  Outfits 
are  supplied  to  physicians  on  request.  These  consist  as  a  rule 


THE  DIPHTHERIA  BACILLUS  185 

of  a  tube  of  freshly  prepared  Loeffler's  serum,  a  tube  containing 
a  sterile  swab  of  absorbent  cotton  firmly  wound  on  a  strong  iron 
wire,  and  printed  directions  and  record  form.  The  whole  outfit 
is  inclosed  in  a  metal  or  wooden  box. 

In  order  to  obtain  a  satisfactory  culture  the  patient  is  placed 
in  a  good  light;  the  tongue  is  depressed  and  the  swab,  removed 
from  its  tube,  is  gently  but  firmly  rubbed  against  any  visible  mem- 
brane without  being  allowed  to  touch  any  other  part  of  the  mouth 
or  throat.  The  swab  is  immediately  inserted  in  the  serum  tube 
and  the  portion  which  has  touched  the  exudate  rubbed  on  the 
surface  of  the  media;  it  is  then  returned  to  its  tube,  the  plug 
inserted,  and  both  tubes  with  the  record  blanks  filled  out  are 
returned  to  the  laboratory. 

When  there  is  no  visible  membrane  it  is  advisable  to  make  two 
cultures,  one  from  the  nose  and  another  from  the  throat.  Need- 
less to  say,  cultures  should  not  be  made  shortly  after  the  applica- 
tion of  a  disinfectant. 

On  reaching  the  laboratory  the  inoculated  tubes  are  placed 
in  an  incubator  at  37°  C.  for  twelve  hours ;  at  the  end  of  that  time 
and  often  before  the  serum  will  be  found  to  be  dotted  with  small 
colonies.  A  microscopic  preparation  is  made  by  first  placing  a 
platinum  loopful  of  sterile  water  upon  a  glass  slide,  and  then  by 
means  of  a  platinum  needle  a  number  of  typical  colonies  are 
removed  from  the  culture  tube  and  smeared  in  the  droplet  of 
water  over  the  slide.  The  preparation  is  fixed  in  the  usual  way 
and  stained  with  Loeffler's  methylene  blue  for  about  five  minutes. 
Examined  with  the  oil  immersion  lens  the  film  may  show  enormous 
numbers  of  diphtheria  bacilli  with  few  cocci,  or  the  reverse,  or  an 
equal  number  of  both  forms. 

An  immediate  diagnosis  can  often  be  made  without  the  use  of 
cultures  by  smearing  a  little  of  the  exudate  from  the  swab  directly 
over  the  slide.  The  result  is  a  little  less  satisfactory ;  the  bacilli 
appear  less  typical  and  are  mixed  with  fibrin  and  epithelial  cells. 

Animal  Inoculations  as  a  Test  of  Toxicity.  —  No  means  of  deter- 
mining with  certainty  the  virulence  of  diphtheria  and  diphtheria- 
like  organisms  found  in  the  throats  of  patients  not  showing  signs 


186  BACTERIOLOGY  FOR  NURSES 

of  diphtheria  or  in  the  throats  of  healthy  individuals  suspected 
of  being  carriers  is  known  save  that  of  animal  inoculation.  For 
this  purpose  an  alkaline  forty-eight-hour  broth  culture  is  employed 
and  two  guinea  pigs  are  inoculated  subcutaneously,  one  with  two 
c.c.  of  the  culture,  the  other  with  the  same  amount  of  culture  plus 
a  protective  amount  of  antitoxin.  If  within  four  days  the  guinea 
pig  receiving  the  toxin  only  dies,  and  the  one  receiving  the  toxin 
plus  antitoxin  lives,  the  organisms  injected  were  undoubtedly 
diphtheria  bacilli. 

Another  and  more  economical  method  is  as  follows :  the  hair 
is  removed  from  the  abdominal  surface  of  two  250  gram  guinea 
pigs,  by  shaving  or  plucking.  The  twenty-four-hours  growth 
on  Loeffler's  serum  of  the  organism  to  be  tested  is  emulsified  with 
20  c.c.  of  salt  solution,  and  0.15  c.c.  of  this  suspension  is  injected 
intracutaneously  into  the  prepared  abdominal  surface  of  each  of 
the  two  guinea  pigs.  One  of  the  animals  is  injected  intracardially 
at  the  same  time  with  250  units  of  antitoxin  or  an  intraperitoneal 
injection  of  the  antitoxin  is  made  twenty-four  hours  before.  In 
this  way  six  cultures  may  be  tested  on  the  same  animals.  Virulent 
diphtheria  bacilli  produce  an  infiltration  and  superficial  necrosis 
at  the  site  of  inoculation  in  from  two  to  three  days,  while  in 
the  guinea  pig  protected  by  the  antitoxin  the  skin  remains 
normal. 

Bacteria  Resembling  Bacillus  Diphtheria.  —  Bacillus  Hoff- 
manni  organisms  often  spoken  of  as  pseudo  diphtheria  bacilli  are 
frequently  found  in  normal  throats  and  in  some  instances  in  those 
of  diphtheritic  individuals.  At  first  they  were  regarded  as  atten- 
uated diphtheria  bacilli.  Later  investigators,  however,  consider 
them  as  a  different  species.  They  appear  as  short,  thick  rods, 
stain  solidly  with  methylene  blue,  do  not  show  granules  when 
stained  with  Neisser's  stain,  are  not  motile,  and  do  not  form  spores. 
Their  colony  growth  on  Loeffler's  serum  media  closely  resembles 
that  of  the  diphtheria  bacillus.  They  differ  from  the  latter  or- 
ganism, however,  in  that  they  are  unable  to  ferment  any  of  the 
sugars;  they  do  not  produce  toxin  and  are  not  pathogenic  for 
guinea  pigs. 


THE  DIPHTHERIA  BACILLUS  187 

Bacillus  Xerosis.  —  Diphtheria-like  bacilli  were  found  by 
Hutschert  and  Neisser  in  1904  in  a  chronic  form  of  conjunctivitis 
known  as  xerosis,  which  they  believed  to  be  the  causal  agent. 
Since  then  the  organism  has  so  frequently  been  isolated  from  nor- 
mal eyes  that  it  is  no  longer  considered  as  the  cause  of  the  disease. 
Morphologically  it  is  almost  identical  with  the  diphtheria  bacillus. 
It  differs  from  B.  diphtherise  and  B.  Hoffmanni  in  its  ability  to 
ferment  sugars,  but  it  resembles  the  latter  in  that  it  produces  no 
toxin  and  is  non-pathogenic  for  animals. 

Still  other  organisms  exist  which  closely  resemble  the  diphtheria 
bacillus  structurally.  They  are  apparently  numerous  and  have 
been  found  both  in  normal  and  diseased  conditions,  although 
it  is  considered  somewhat  doubtful  whether  they  ever  incite  dis- 
ease. As  yet  no  classification  of  these  organisms  has  been  made 
and  they  are  grouped  together  under  the  term  Diphtheroids. 


CHAPTER  XVIII 

THE  TUBERCLE  BACILLUS  AND   OTHER   ACID-FAST 

ORGANISMS 

IT  is  estimated  that  in  the  United  States  160,000  persons 
die  each  year  of  tuberculosis.  For  centuries  the  disease  has 
been  recognized,  but  only  within  comparatively  recent  times 
has  its  infectiousness  been  scientifically  established.  The  fact 
that  tuberculosis  might  be  induced  by  inoculation  with  tuber- 
culous material  was  demonstrated  by  Villemin  in  1865.  Baum- 
garten  early  in  1882  described  the  bacilli  in  tissue  sections,  but  it 

remained  for  Robert  Koch  to 
isolate  the  organism,  to  grow 
it  in  pure  culture,  and  with  the 
pure  culture  to  reproduce  the 
characteristic  lesions  in  ani- 
mals. Koch  announced  his 
discovery  in  1882  and  in  1884 
he  submitted  his  full  report. 

Morphology  and  Staining. 
—  Tubercle  bacilli  appear  as 
slender,  non-motile  rods  about 
2  p  to  4  p  in  length  and  0.3 

FIG.  28.  —  Tubercle  Bacilli.  i\  r        •          -1,1         T  i         i 

to  0.5  /*  m  'width.     In  colored 

preparations  they  may  appear  straight  or  slightly  curved,  single 
or  lying  together  in  heaps.  Ordinarily  they  stain  uniformly  (Fig. 
28).  Frequently  however,  deeply  stained  thickenings  are  seen 
which  give  to  the  organism  a  somewhat  beaded  appearance.  At 
first  these  granules  were  thought  to  be  spores.  Soon,  however, 
it  was  shown  that  the  bacilli  containing  them  were  no  more  re- 

188 


THE  TUBERCLE  BACILLUS  189 

sistant  to  heat  and  drying  than  others  in  which  they  were  not 
found  and  that  consequently  they  could  not  be  regarded  as  spores. 

Occasionally  long  thread-like  branching  forms,  sometimes  with 
swollen  ends,  are  seen.  They  are  considered  by  some  observ- 
ers as  involution  forms;  others  regard  them  as  normal  and  on 
this  basis  class  the  tubercle  bacilli  either  with  the  higher  bacteria 
(trichomycetes)  or  with  the  true  molds;  still  others  place  them 
and  closely  related  forms  in  a  separate  group  intermediate  between 
the  ordinary  bacteria  and  the  higher  forms.  Their  classification 
is  still  an  unsettled  question. 

Another  peculiarity  of  the  tubercle  bacillus  is  the  possession  of 
a  waxy  envelope,  which  not  only  confers  upon  it  an  additional 
degree  of  resistance,  but  which  also  prevents  it  from  readily  taking 
up  the  ordinary  anilin  dyes.  Once  stained,  however,  it  is  with  diffi- 
culty that  the  color  can  be  removed  even  by  the  use  of  strong 
acids.  The  property  is  so  characteristic  that  the  tubercle  bacillus 
and  certain  other  closely  related  organisms  are  termed  "  acid- 
fast."  This  staining  peculiarity  makes  it  possible  to  recognize 
the  organism  immediately  in  film  preparations  from  pus,  sputum, 
etc.  Details  of  the  special  stains  and  methods  employed  have  al- 
ready been  described. 

Cultivation.  —  On  account  of  their  slow  growth  tubercle  bacilli 
are  difficult  to  cultivate  and  even  more  difficult  to  isolate.  Koch 
succeeded  in  obtaining  a  pure  culture  by  carefully  rubbing  tuber- 
culous tissue  over  the  surface  of  coagulated  beef  serum,  and  then 
after  about  two  weeks'  incubation  there  appeared  minute  points  of 
irregular  whitish  growth  which  he  compared  to  small,  dry  scales. 
Once  isolated  the  organism  will  grow  readily  on  egg  medium  or 
on  agar  containing  3  to  5  per  cent  glycerin ;  in  from  ten  to  fourteen 
days  growth  appears  as  a  dull,  whitish,  wrinkled  film.  In  thin 
layers  of  glycerin  broth,  if  a  small  amount  of  growth  is  carefully 
placed  on  the  surface  it  spreads  as  a  wrinkled  pellicle  from  one 
side  of  the  container  to  the  other;  this  method  of  cultivation  is 
usually  employed  for  the  production  of  tuberculin. 

It  frequently  happens  that  in  tuberculous  tissue  tubercle  bacilli 
are  found  free  from  contaminating  organisms  and  a  practically 


190  BACTERIOLOGY  FOR  NURSES 

pure  culture  is  obtained  on  media  inoculated  with  such  tissue. 
Sputum,  on  the  contrary,  usually  contains  many  other  varieties 
which  grow  with  much  greater  facility  than  the  tubercle  bacillus 
and  so  completely  inhibit  the  growth  of  the  latter.  Isolation  from 
such  material  is  best  accomplished  by  injecting  it  into  guinea 
pigs ;  the  animal  will  die  in  from  four  to  six  weeks  and  the  bacilli 
may  be  obtained  in  pure  culture  from  the  lymph  nodes  near  the 
point  of  injection  and  frequently  from  tubercles  in  the  various 
organs.  The  optimum  temperature  for  growth  is  37°  to  38°  C. ; 
below  30°  and  above  42°  C.  development  rarely  occurs. 

Resistance.  —  Tubercle  bacilli  show  a  greater  degree  of  resist- 
ance to  external  influences  than  most  non-spore-bearing  organisms. 
When  completely  dried  they  can  withstand  a  temperature  of 
100°  C.  for  forty-five  minutes ;  separated  in  fluids  such  as  milk 
they  are  destroyed  by  exposures  to  60°  C.  in  twenty  minutes. 
Cold  has  little  effect  upon  them.  In  sputum  exposed  to  direct 
sunlight  the  organisms  are  killed  in  a  few  hours ;  in  diffuse  daylight 
in  a  few  days.  Dried  in  rooms  that  have  little  light,  they  have  been 
found  alive  after  ten  months.  It  is  not  probable  that  the  tubercle 
bacilli  ever  multiply  outside  of  the  body  save  in  freshly  expecto- 
rated sputum  and  on  artificial  culture  media.  In  sputum  they  are 
destroyed  in  six  hours  by  the  addition  of  an  equal  quantity  of  5 
per  cent  carbolic  acid.  Bichloride  of  mercury  is  unsatisfactory 
as  a  disinfectant  because  it  combines  with  the  mucus  present  and 
has  little  effect  upon  the  bacteria. 

Pathogenesis.  —  Tubercle  bacilli  do  not  produce  true  toxins, 
but  their  bodies  contain  poisonous  substances,  probably  of  the 
nature  of  endotoxins.  In  the  animal  body  the  local  lesion  pro- 
duced is  usually  in  the  form  of  a  tubercle  or  nodule  which  varies 
somewhat  in  the  different  tissues.  If  the  bacilli  gain  entrance 
to  the  connective  tissue  their  first  action  appears  to  be  on  the  con- 
nective tissue  cells  which  soon  begin  to  show  that  some  irritant 
is  acting  upon  them.  The  cells  become  swollen  and  mitotic  divi- 
sion occurs,  the  resulting  so-called  epitheloid  cells  being  much 
larger  than  the  parent  cell  and  possessing  paler  nuclei.  Very  soon 
small  foci  of  epitheloid  cells  are  formed  about  the  bacilli  and  at 


THE  TUBERCLE  BACILLUS  191 

the  same  time  numbers  of  leukocytes  begin  to  appear  in  the  neigh- 
borhood. When  living  bacilli  are  present  and  sufficiently  viru- 
lent to  multiply  the  lesion  increases,  the  central  cells  degenerate 
into  a  cheese-like  mass,  and  later  a  cavity  results. 

The  most  characteristic  lesions  caused  by  the  tubercle  bacilli 
are  the  so-called  miliary  tubercles  which,  before  they  undergo 
degeneration,  appear  as  hard,  gray,  translucent  nodules  rather 
smaller  than  a  millet  seed  in  size.  Instead  of  the  miliary  tuber- 
cles, however,  a  diffuse  growth  of  tissue  may  occur  similar  in  struc- 
ture to  the  former  and  which  also  tends  to  undergo  cheesy  degen- 
eration. 

The  general  symptoms  of  tuberculosis,  —  fever,  perspiration,  and 
emaciation  —  are  due  to  the  absorption  and  distribution  throughout 
the  body  of  the  bacterial  poison. 

Modes  of  Infection.  —  Occasionally  the  organisms  attack  the 
abraded  skin  or  mucous  membranes  and  lupus  develops  or  a 
nodular  growth.  Their  main  entrance  to  the  body,  however,  is 
through  the  respiratory  tract  or  the  digestive  tract. 

The  organisms  leave  the  body  chiefly  in  the  sputum  of  open 
cases  of  pulmonary  tuberculosis  and  in  other  cases  in  any  dis- 
charges from  tuberculous  lesions  opening  into  the  skin.  In  pul- 
monary cases  the  sputum  is  often  swallowed,  with  the  result  that 
tubercle  bacilli  may  be  excreted  in  the  feces.  Thus  all  of  the  dis- 
charges from  the  body  may  be  infective. 

Because  pulmonary  tuberculosis  is  of  far  more  frequent  occur- 
rence than  any  other  form,  tuberculosis  was  for  a  long  period 
considered  as  an  air-borne  infection.  The  opinion  was  strongly 
expressed  by  Koch  in  1884  and  was  for  many  years  practically 
universally  accepted.  An  interesting  point  in  support  of  this 
theory  is  that  it  requires  very  few  organisms  by  inhalation  to  give 
rise  to  the  disease,  whereas  thousands  are  necessary  by  mouth  to 
produce  infection  of  the  alimentary  canal.  On  the  other  hand, 
the  lungs  are  greatly  protected  from  external  infection  both  by 
their  location  and  by  the  moist  ciliated  epithelium  lining  the  nasal 
and  pharyngeal  passage.  Also  the  fact  that  the  lesion  in  the 
lungs  is  usually  at  the  apex  and  not  in  the  direct  line  that  floating 


192  BACTERIOLOGY  FOR  NURSES 

particles  in  the  air  would  be  mechanically  carried  seems  to  indicate 
that  tubercle  bacilli  may  find  their  way  to  the  lung  tissue  by  other 
means  than  direct  inhalation.  Further,  it  has  been  demonstrated 
that  sputum  exposed  to  direct  sunlight  will,  especially  in  summer, 
be  disinfected  by  the  time  it  is  in  a  condition  to  be  carried  into  the 
air  as  dust.  Tuberculous  sputum  when  expectorated  in  shady  parts 
of  the  street,  or  in  houses,  or  in  dark  places,  however,  constitutes 
a  real  menace.  It  has  been  estimated  that  as  many  as  five  billion 
tubercle  bacilli  may  be  expectorated  by  a  single  individual  in 
twenty-four  hours.  Consequently  the  neighborhood  of  tuberculous 
individuals  who  expectorate  without  taking  any  precautions  to 
prevent  the  spread  of  infection  is  exceedingly  dangerous.  In 
rooms  occupied  by  such  persons  dried  sputum  containing  virulent 
bacilli  may  be  constantly  in  the  air  blown  about  by  sweeping, 
walking,  the  closing  of  doors,  etc.  As  long  as  sputum  remains 
moist  there  is  no  danger  of  infection  by  inhalation;  the  only 
danger  then  lies  in  direct  contact. 

During  ordinary  breathing  the  expirations  of  a  patient  suffer- 
ing from  pulmonary  tuberculosis  are  normally  free  from  bacteria. 
In  forced  efforts,  however,  such  as  coughing,  sneezing,  and  loud 
speaking,  fine  particles  of  throat  secretion  are  thrown  out  as  a  light 
spray,  which  may  be  laden  with  organisms  that  may  have  been 
present  in  the  mouth.  Tubercle  bacilli  thus  sprayed  may  fall 
directly  on  the  mucous  membrane  of  a  healthy  individual  or  may 
be  conveyed  indirectly  by  food  or  other  objects. 

As  early  as  1868,  and  many  years  before  the  discovery  of  the 
tubercle  bacillus,  Chauveau  suggested  that  the  causal  agent  might 
gain  entrance  by  way  of  the  intestinal  canal.  Later  investigators 
have  proved  beyond  question  that  such  often  is  the  case.  Tuber- 
cle bacilli  ingested  in  food  and  drink  may  pass  through  the  mucous 
membrane  of  the  digestive  tube  without  leaving  any  trace  of 
their  passage,  gain  access  to  the  blood,  and  so  be  carried  to  dis- 
tant parts  of  the  body.  The  fact  that  the  disease  usually  localizes 
itself  in  the  lungs  may  be  because  this  organ  presents  the  least 
resistance.  It  is  even  claimed  by  some  authorities  that  no  matter 
how  the  tubercle  bacillus  reaches  an  individual,  whether  by  dust 


THE  TUBERCLE  BACILLUS  193 

or  droplets,  fingers  or  food,  it  passes  either  through  the  tonsils 
or  the  mucous  membranes  of  the  upper  respiratory  tract  or  is 
carried  to  the  intestines  and  passed  through  the  tissues  there. 

Infection  by  drinking  milk  from  tuberculous  cows  has  been 
clearly  demonstrated.  It  has  also  been  shown  that  such  infection 
does  not  necessarily  come  from  cows  with  tuberculous  lesions  of 
the  udder,  but  may  be  conveyed  in  milk  from  cows  showing  no 
lesions  of  the  udder  whatever.  Perhaps  in  all  such  cases  dried 
feces  falling  into  the  milk  from  the  skin  of  the  cow  is  responsible 
for  the  presence  of  the  tubercle  bacillus.  Human  infection  by 
this  means  must  necessarily  pass  by  way  of  the  tonsils  or  the  ali- 
mentary tract.  The  majority  of  cases  of  cervical  adenitis  and 
abdominal  tuberculous  in  young  children  are  undoubtedly  con- 
tracted in  this  manner. 

It  may  be  stated,  then,  that  the  two  chief  modes  of  infection  are 
by  inhalation  and  by  ingestion  of  the  tubercle  bacillus.  In  the 
former  the  organisms  are  for  the  most  part  derived  from  human 
beings;  in  the  latter,  milk  and  milk  products  from  tuberculous 
cows  or  food  contaminated  from  human  cases  are  responsible. 

Heredity. —  In  the  strict  sense  of  the  word  tuberculosis  is  not 
considered  hereditary.  It  is  extremely  unlikely  that  spermatozoa 
or  ova  infected  by  tubercle  bacilli  would  undergo  normal  develop- 
ment. It  is  generally  conceded  that  a  hereditary  tendency  or 
disposition  to  the  disease  may  be  transmitted  from  the  parent 
to  the  offspring,  although  what  the  tendency  is  has  not  been  clearly 
defined ;  it  may  be  a  feeble  constitution,  or  a  structural  peculiarity 
or  possibly  an  inability  on  the  part  of  the  body  cells  to  generate 
defensive  antibodies  when  infection  occurs.  Congenital  infection, 
though  rare,  does  occasionally  occur,  in  which  case  tubercle  bacilli 
pass  from  the  mother  to  the  fetus  by  way  of  the  placenta.  Ex- 
trauterine  infection  is  much  more  likely  to  be  the  cause  of  tuber- 
culosis in  infants.  Animal  experiments  have  shown  that  the  young 
of  infected  mothers  are  usually  infected  only  when  suckled  by  the 
tuberculous  parent;  when  nourished  by  a  healthy  foster  mother 
they  remain  normal.  The  fact  that  tuberculosis  seems  to  persist 
in  certain  families  may  be  due  solely  to  the  intimate  associations 


194  BACTERIOLOGY  FOR  NURSES 

of  home  life  and  the  lack  of  precautions  in  preventing  the  sick 
from  infecting  the  well. 

Immunity.  —  Although  recovery  from  tuberculosis  is  of  fre- 
quent occurrence  the  processes  involved  are  extremely  obscure. 
Many  attempts  have  been  made  to  produce  artificial  immunity  in 
tuberculosis  as  in  other  infectious  diseases,  but  so  far  all  have 
failed.  Tuberculous  infection  appears  to  differ  in  many  respects 
from  other  infectious  processes.  Tubercle  bacilli  may  invade 
the  tissues  and  foci  develop  without  sufficient  bodily  disturbance 
to  attract  attention ;  also  an  infection,  so  far  as  clinical  symptoms 
are  concerned,  may  be  completely  cured,  yet  the  focus  may  remain 
and  though  completely  walled  off  and  non-progressive  may  con- 
tain virulent  tubercle  bacilli  during  the  whole  of  the  individual's 
life. 

Koch  discovered  that  infected  animals  reacted  differently  to 
an  injection  of  living  bacilli  than  did  normal  animals.  When 
healthy  animals  are  inoculated  with  virulent  organisms  tubercles 
develop  near  the  point  of  inoculation;  the  infection  is  usually 
carried  to  the  various  organs  and  the  animal  dies  of  generalized 
tuberculosis.  A  tuberculous  animal  on  the  contrary  shows  an 
immediate  and  violent  reaction.  A  marked  inflammatory  area 
around  the  point  of  injection  occurs,  followed  sometimes  by  necrosis 
and  sloughing,  but  with  no  advance  of  the  infection  beyond  the 
point  of  injection.  The  reaction  well  illustrates  the  phenomenon 
of  hypersusceptibility.  Following  a  first  injection  of  the  tubercle 
bacilli  or  infection  by  other  means  the  tissue  cells  offer  no  imme- 
diate resistance  and  the  disease  progresses.  The  presence  of  the 
organisms,  however,  so  sensitizes  the  cells  that  a  second  invasion 
is  resisted  immediately  and  vigorously,  and  protecting  substances 
and  phagocytic  cells  are  concentrated  upon  the  point  where  they 
are  most  needed,  as  is  evidenced  by  the  prompt  inflammatory 
reaction. 

Koch,  as  a  result  of  these  observations,  concluded  that  the 
resistance  of  tuberculous  individuals  might  be  further  increased 
by  the  injection  of  disintegrated  bacteria  and  the  products  of 
their  growth,  and  with  this  in  view  he  prepared  tuberculin.  Un- 


THE  TUBERCLE  BACILLUS  195 

fortunately  the  great  hopes  at  first  entertained  have  not  been 
realized.  A  certain  number  of  cases  of  increased  resistance  and 
clinical  cures  have,  however,  been  reported  from  its  use  as  a 
therapeutic  agent. 

The  tuberculin  reaction  is  local,  focal,  and  general.  The  local 
reaction  appears  as  an  inflammatory  condition  at  the  point  of 
inoculation.  The  focal  reaction  consists  of  an  increased  blood 
supply  around  the  infected  area  and  a  consequent  softening  of 
the  focus  and  a  liberation  of  toxic  products  which  give  rise  to  the 
general  reaction. 

If  the  dose  of  tuberculin  is  not  too  large  the  focal  reaction  soon 
subsides,  with  the  result  that  increased  cellular  activity  has  caused 
a  further  proliferation  of  connective  tissue  and  fortified  the  wall 
surrounding  the  tuberculous  process.  In  chronic  lesions  of  the 
bones  or  inactive  skin  or  ear  cases,  in  which  the  body  cells  are  only 
feebly  active  in  self-defense,  small  doses  of  tuberculin  are  reported 
to  have  a  stimulating  beneficial  effect.  Should  the  dose  of  tuber- 
culin be  too  large  or  the  body  cells  incapable  of  reacting,  the  focal 
reaction  may  be  a  softening  and  breaking  down  of  the  lesion  with 
liberation  of  the  bacilli  and  spread  of  the  tuberculous  area.  Thus 
tuberculin  as  a  therapeutic  agent  is  a  somewhat  dangerous  weapon. 
Its  success  appears  to  rest  upon  administering  just  the  right  amount 
to  call  forth  sufficient  response  without  overtaxing  the  already 
sensitized  body  cells. 

Tuberculin  as  a  Diagnostic  Agent.  —  The  allergic  or  hypersen- 
sitive state  of  the  tissue  cells  in  tuberculous  individuals  makes 
possible  the  use  of  tuberculin  as  a  diagnostic  agent,  although  the 
phenomenon  is  as  yet  little  understood. 

A  number  of  tuberculin  preparations  have  been  employed. 
The  following  is  the  method  originally  employed  by  Koch  and 
usually  designated  "  O.T."  A  six-weeks-old  culture  of  tubercle 
bacilli  in  5  per  cent  glycerin  broth  is  killed  by  heat,  filtered,  and 
evaporated  down  to  one  tenth  of  its  original  volume.  The  resulting 
fluid  thus  contains  the  products  of  disintegrated  bacilli,  substances 
formed  from  the  medium  during  their  growth  and  the  medium 
itself. 


196  BACTERIOLOGY  FOR  NURSES 

The  Intracutaneous  Test  of  Mantoux.  —  The  test  is  made  as 
follows :  the  skin  of  the  forearm  is  cleansed  with  alcohol  and  then 
with  ether  and  a  series  of  injections  of  different  dilutions  of  tuber- 
culin are  made  intracutaneously.  Four  dilutions  are  used :  1  to 
10,000,000,  1  to  1,000,000,  1  to  100,000,  and  1  to  10,000.  The 
amount  injected  of  each  is  generally  0.1  c.c.  A  control  injec- 
tion of  0.1  c.c.  of  sterile  normal  salt  solution  is  made  at  the  same 
time.  A  positive  reaction  appears  in  six  or  eight  hours  and  usually 
subsides  in  six  to  ten  days. 

The  Percutaneous  Test  of  Moro.  —  Equal  parts  of  lanolin  and 
tuberculin  are  made  into  an  ointment  and  a  small  amount  is  rubbed 
into  the  skin  on  the  chest.  A  positive  reaction  appears  within 
one  to  four  days  as  an  eruption  of  slightly  elevated  papules. 

The  Cutaneous  Test  of  Von  Pirquet.  —  The  test  is  carried  out 
as  follows :  the  forearm  is  cleansed  with  alcohol  and  ether  and 
two  small  scratches  are  made  about  three  inches  apart,  care  being 
taken  not  to  cause  bleeding.  On  one  scratch  a  drop  of  tuber- 
culin is  placed,  the  other  is  left  as  a  control.  A  positive  reaction 
may  appear  in  from  three  to  ten  hours  as  a  slightly  raised  redden- 
ing of  the  skin,  usually  circular  and  about  10  mm.  in  diameter. 

Ophthalmic  Test  of  Calmette.  —  For  this  test  either  a  2  per 
cent  solution  of  Koch's  old  tuberculin  or  a  purified  form  is  used. 
One  drop  of  the  solution  is  placed  in  the  conjunctival  sac  and  the 
fluid  is  allowed  to  spread  over  the  surface.  In  a  positive  reaction 
the  conjunctiva  is  inflamed.  The  lids  become  congested  and  their 
inner  surface  of  a  bright  red  color,  and  varying  amounts  of  a 
fibrinous  exudate  appears.  The  reaction  reaches  its  maximum 
in  from  six  to  ten  hours  and  disappears  usually  in  from  two  to 
three  days.  The  ophthalmic  test  is  easily  applied,  but  is  little 
employed,  owing  to  serious  dangers  that  may  result. 

So  far  as  is  known  there  is  no  positive  skin  reaction  without 
infection.  A  positive  reaction,  however,  tells  nothing  of  the 
location  or  extent  of  the  lesion  nor  if  it  is  a  progressive  or  an  en- 
capsulated focus.  Very  advanced  cases  frequently  show  little 
response  to  tuberculin  tests,  the  tissue  cells  being  evidently  in- 
capable of  further  effort. 


THE  TUBERCLE  BACILLUS  197 

Varieties  of  Tubercle  Bacilli.  —  At  least  four  types  of  tubercle 
bacilli  are  recognized :  human,  bovine,  avian,  and  fish.  The  human 
and  bovine  varieties  closely  resemble  each  other.  The  former 
appear  somewhat  longer  and  more  slender  than  the  latter,  show  a 
greater  tendency  to  irregularities  in  staining,  and  grow  more 
luxuriantly  on  culture  media.  The  important  difference  lies  in 
the  fact  that  the  human  type  is  very  pathogenic  for  man,  but  is 
considerably  less  so  for  cattle  and  other  animals.  On  the  other 
hand,  the  bovine  type  is  very  pathogenic  for  almost  all  mammalians 
except  man ;  it  is  pathogenic  for  man,  but  much  less  so  than  the 
human  type.  The  critical  laboratory  test  for  differentiating  the 
two  varieties  is  made  on  rabbits ;  a  one  hundredth  of  a  gram  of 
a  young  bovine  culture  injected  intravenously  into  a  rabbit  will 
cause  generalized  tuberculosis  in  about  six  weeks,  whereas  fifty 
to  one  hundred  times  the  amount  of  the  human  variety  produces 
at  most  a  slight  tuberculous  lesion. 

About  10  per  cent  of  all  cases  of  tuberculosis  in  young  children 
under  five  years  of  age  is  due  to  bovine  tubercle  bacilli.  The  fact 
that  such  infections  are  usually  localized  in  the  cervical  or  abdom- 
inal lymph  nodes  strongly  suggest  that  the  portal  of  entry  is  the 
tonsils  or  small  intestines  and  that  cows'  milk  is  the  source  of 
origin. 

Avian  tubercle  bacilli  correspond  in  morphology  and  staining 
reactions  with  the  above  types.  They  differ  in  that  they  grow 
luxuriantly  on  culture  media  at  45°  C.  and  can  even  multiply  at 
a  temperature  as  high  as  50°  C.  On  glycerin  agar  or  blood 
serum  an  abundant  growth  appears  within  ten  days,  white, 
moist,  and  fat-like,  and  totally  different  to  the  dried  and 
wrinkled  appearance  of  the  human  type.  Chickens,  pigeons, 
and  pheasants  are  very  susceptible ;  geese  and  ducks  appear  to 
be  immune. 

The  tubercle  bacillus  of  fish  was  first  isolated  from  lesions  in 
a  carp.  Microscopically  it  resembles  the  other  forms.  On  culture 
media  growth  is  thick  and  moist  like  that  of  the  avian  type.  Its 
temperature  requirements,  however,  are  very  different  to  the  vari- 
eties found  in  warm-blooded  animals;  growth  occurs  between 


198  BACTERIOLOGY  FOR  NURSES 

12°  and  36°  C.,  the  optimum  temperature  being  25°  C.     Neither 
the  avian  nor  the  fish  tubercle  bacilli  are  pathogenic  for  man. 


OTHER  ACID-FAST  BACILLI 

Bacillus  of  Leprosy.  —  Hansen  in  1874  reported  the  presence 
of  a  bacillus  in  the  tubercles  of  leprous  individuals  which  somewhat 
resembled  the  tubercle  bacillus.  Later  many  other  observers  con- 
firmed Hansen's  report.  In  tissue  sections  the  bacilli  appear  as 
thin  rods  generally  within  the  cells  of  the  granulation  tissue  and 
are  often  so  numerous  that  the  cell  structure  is  hidden ;  usually 
they  are  arranged  parallel  to  one  another  and  present  the  appear- 
ance of  small  bundles.  They  take  up  the  basic  anilin  dyes  more 
readily  than  the  tubercle  bacilli,  but  like  them  they  resist  decolori- 
zation  with  the  mineral  acids  and  alcohols.  The  organisms 
differ  from  the  tubercle  bacilli  in  that  they  grow  with  difficulty 
on  artificial  culture  media  and  are  much  less  if  at  all  pathogenic 
for  the  lower  animals. 

In  1908  certain  workers  succeeded  in  growing  an  acid-fast 
organism  on  plain  agar  in  symbiosis  with  ameba  and  other  bacteria, 
and  then  by  killing  the  other  organisms  by  means  of  heat  they 
obtained  a  pure  culture  of  "  acid-fast "  bacilli.  There  is  no  satis- 
factory evidence,  however,  that  such  organisms  will  reproduce 
the  disease  in  experimental  animals. 

The  negative  results  following  attempts  to  produce  the  disease 
by  inoculations  of  leprous  tissue  have  led  to  the  assumption  that 
the  bacilli  in  such  tissue  must  be  for  the  most  part  dead.  An 
apparently  successful  attempt  was  made  upon  a  criminal  in  the 
Sandwich  Islands,  who  obtained  pardon  on  condition  that  he  allow 
himself  to  be  inoculated  with  leprosy.  He  consented  and  the 
disease  did  develop  two  or  three  years  later.  The  experiment  is 
open  to  objection,  however,  because  the  man  had,  before  inocula- 
tion, been  frequently  in  contact  with  lepers  and  had  thus  been 
exposed  to  the  infection  in  a  natural  way. 

It  has  been  supposed  by  some  observers  that  leprosy  is  a  form 
of  tuberculosis.  There  is  little  ground  for  the  supposition,  although 


OTHER  ACID-FAST  ORGANISMS  199 

it  has  been  found  that  a  considerable  portion  of  lepers  react  to 
tuberculin  like  tuberculous  patients. 

Rat  Leprosy.  —  A  disease  occurs  in  rats  which  closely  resembles 
leprosy.  The  relation  between  the  disease  and  that  occurring 
in  human  beings  has  not  as  yet  been  established. 

Smegma  Bacillus.  —  The  organism  is  present  in  the  secretions 
of  the  external  genitals.  It  has  much  the  same  appearance  and 
staining  reaction  as  the  tubercle  bacillus.  It  is  non-pathogenic. 

Bacillus  of  Johne's  Disease  or  Paratubercular  Dysentery  of 
Cattle.  —  The  disease  is  characterized  by  chronic  diarrhea  and 
emaciation.  The  organisms  isolated  from  the  lesions  closely  re- 
semble the  tubercle  bacillus. 

Timothy  Grass  Bacillus.  —  Other  organisms  showing  various 
degrees  of  acid-fastness  have  been  isolated  from  grass,  cow  manure, 
milk,  butter,  etc.  They  have  little  interest  apart  from  the  fact 
that  they  can  only  be  distinguished  from  the  tubercle  bacillus  by 
animal  inoculation. 


CHAPTER  XIX 

INTESTINAL  BACTERIA.    THE   COLON-TYPHOID 

GROUP 

DURING  life  a  great  variety  of  organisms  find  suitable  conditions 
for  development  in  the  intestinal  tract.  At  birth  the  meconium 
of  healthy  infants  is  sterile.  Very  soon,  however,  bacteria  gain 
entrance  to  the  alimentary  canal  through  the  rectum  or  by  way 
of  the  mouth  from  swallowing  saliva  or  food.  As  the  reaction 
and  amount  of  oxygen  at  different  levels  vary,  each  species  tends 
to  remain  in  that  portion  of  the  tract  in  which  it  finds  its  optimum 
conditions.  Aerobic  organisms  are  most  abundant  in  the  mouth, 
although  hidden  in  the  crypts  of  the  tonsils  and  in  folds  of  mucous 
membrane  anaerobes  may  flourish.  Those  organisms  which  are 
able  to  withstand  the  acidity  of  the  gastric  juice  and  succeed  in 
reaching  the  duodenum  find  the  alkaline  reaction  there  much 
more  favorable  to  development;  also  the  diminished  amount  of 
oxygen  renders  conditions  particularly  suitable  for  anaerobic  or 
facultative  anaerobic  growth.  The  available  food  supply  is 
probably  the  most  important  factor  in  determining  the  type  of 
organisms  likely  to  develop.  In  breast-fed  infants,  for  example, 
lactose  being  more  abundant  than  any  other  ingredient  in  the 
milk,  fermentative  organisms  predominate.  Later  as  protein  is 
added  to  the  diet  proteolytic  bacteria  become  more  numerous 
and  the  fermentative  type  relatively  decrease.  Many  investiga- 
tors have  tried  to  determine  whether  the  presence  of  bacteria  in 
the  intestinal  tract  is  of  physiological  benefit  to  the  individual. 
Successful  experiments  have  shown  that  at  least  they  are  not  a 
necessity.  An  infection  with  pathogenic  organisms,  such  as 
dysentery  or  cholera,  totally  changes  for  a  time  intestinal  condi- 

200 


INTESTINAL  BACTERIA  201 

tions.  The  invaders  multiply,  produce  local  lesions,  and  more 
or  less  crowd  out  the  normal  inhabitants. 

Apart  from  infections  abnormal  conditions  may  result  from 
the  unbalanced  activities  of  the  organisms  ordinarily  present  in 
the  intestines.  So  far  as  at  present  known  two  distinct  processes 
may  be  concerned :  (1)  excessive  bacterial  proteolysis,  by  means 
of  which  toxic  substances  are  produced  from  the  protein  ingested 
as  food  which  when  absorbed  by  the  body  cells  give  rise  to  the 
condition  known  as  "  auto-intoxication,"  or  (2)  excessive  carbo- 
hydrate fermentation  which  may  result  in  an  overproduction  of 
acids  or  other  irritating  substances  and  cause  a  chronic  diarrheal 
condition. 

Of  the  many  varieties  of  bacteria  occupying  the  intestines  of 
man  and  animals  a  certain  group,  ordinarily  non-pathogenic,  are 
classed  together  as  "  colon  bacilli  "  because  they  live  in  the  colon 
and  have  similar  characteristics.  Closely  related  morphologically 
and  biologically  to  the  colon  bacilli  are  a  number  of  other  organ- 
isms which  when  they  gain  access  to  the  intestines  give  rise  to 
distinctly  morbid  conditions.  The  entire  group  is  termed  the 
colon-typhoid  group.  Such  members  as  the  typhoid  and  para- 
typhoid bacilli,  including  the  types  responsible  for  meat  poisoning, 
are  specifically  pathogenic ;  the  colon  bacilli  and  their  near  rela- 
tives are  pathogenic  only  under  certain  circumstances. 

The  group  is  usually  arranged  in  four  subdivisions : 

1.  Colon  Group.     Normally  present  in  the  intestines,  rarely 

pathogenic. 

2.  Para-typhoid  Group.     Possessing  varying  degrees  of  patho- 

genicity. 

3.  Bacillus  typhosus.    Pathogenic. 

4.  Dysentery  Group.     Pathogenic. 

All  members  of  the  entire  group  possess  certain  common  char- 
acteristics. They  are  rather  short,  non-spore-bearing  bacilli ;  stained 
by  Gram's  method  they  are  decolorized;  none  of  them  liquefies 
gelatin. 

Apart  from  these  general  features  there  appears  to  be  a  variety 


202  BACTERIOLOGY   FOR  NURSES 

of  cross  relationships,  and  in  order  to  determine  the  subdivisions 
and  to  differentiate  the  members  of  each  subdivision  careful 
observation  of  their  fermentative  action  on  the  various  carbo- 
hydrate media,  and  their  agglutinative  reaction  in  immune  sera 
is  necessary. 

From  the  above  it  will  be  understood  that  around  each  par- 
ticular type  a  number  of  variants  are  grouped.  Thus,  for  example, 
in  different  epidemics  of  dysentery  different  strains  of  bacilli 
have  been  isolated,  varying  from  each  other  only  in  minor  details, 
but  corresponding  in  certain  points  which  mark  them  as  close 
relatives  and  warrant  their  classification  as  one  group. 

The  Colon  Group.  —  The  first  description  of  a  member  of  this 
group  was  given  by  Emmerich  in  1885,  who  isolated  the  organism 
from  the  dejecta  of  a  patient  suffering  from  Asiatic  cholera.  In 
1886  Escherich  obtained  a  similar  bacillus  from  the  feces  of  healthy 
infants,  to  which  he  gave  the  name  Bacillus  coli  communis.  Later 
it  was  shown  that  closely  allied  types  are  normal  inhabitants  of 
man  and  animals. 

The  organisms  are  widely  distributed  in  nature.  Transferred 
through  the  feces  as  manure  or  sewage,  they  are  found  on  culti- 
vated land  and  in  surface  waters.  They  are  most  abundant, 
however,  in  the  intestines  of  man  and  animals  and  particularly 
in  that  portion  from  which  they  derive  their  name.  Apart  from 
the  fact  that  under  certain  conditions  the  organisms  may  excite 
disease,  they  have  special  hygienic  interest  in  that  their  presence 
in  water  or  milk  is  an  indication  of  fecal  pollution.  The  presence 
of  colon  bacilli  does  not  necessarily  mean  the  presence  of  typhoid 
or  dysentery  bacilli,  but  it  indicates  the  possibility  when  the 
contamination  is  of  human  origin. 

B.  Coli  Communis.  —  The  colon  group  contains  many  varieties ; 
of  these  B.  coli  communis  has  probably  been  the  most  studied 
and  may  be  considered  as  the  most  representative. 

Morphology  and  Staining.  —  The  typical  forms  appear  as 
short  rods  with  rounded  ends  ranging  from  1  to  3  /*  in  length 
and  0.4  to  0.7  /*  in  diameter.  They  possess  seven  or  eight 
peritrichic  flagella.  Motility,  however,  varies  in  the  different 


COLON  GROUP  r  203 

strains;  sometimes  in  young  cultures  it  is  quite  active;  in 
others  it  may  be  so  sluggish  as  to  be  hardly  distinguishable  from 
Brownian  movement.  B.  coli  communis  stains  readily  with  the 
ordinary  aniline  dyes,  is  Gram  negative,  and  does  not  form  spores. 

Cultivation.  —  The  organism  is  an  aerobic  and  facultative 
anaerobe.  It  grows  best  at  37°  C.,  but  multiplication  will  occur 
as  low  as  10°  C.  It  develops  on  the  simplest  culture  media;  in 
broth  it  grows  rapidly  causing  a  general  clouding  of  the  medium. 
In  gelatin  stabs  growth  occurs  along  the  line  of  inoculation  and 
spreads  along  the  surface  of  the  medium  almost  to  the  sides  of 
the  tube.  Surface  colonies  on  agar  are  of  a  grayish  color,  round 
and  glistening,  and  often  showing  a  peculiar  structure  somewhat 
resembling  a  grape  leaf.  Colonies  growing  deep  in  the  medium 
may  be  oval  or  the  shape  of  a  whetstone.  On  potato  growth  is 
abundant,  changing  from  a  grayish  white  in  young  cultures  to  a 
yellowish  brown  in  older  ones.  In  milk  coagulation  occurs  from 
one  to  four  days,  principally  due  to  the  production  of  lactic  and 
acetic  acid  from  the  lactose  present.  In  lactose-litmus-agar  the 
medium  becomes  red  and  gas  bubbles  frequently  appear  by  the 
side  or  under  the  colonies.  The  organism  is  able  to  ferment  a 
number  of  carbohydrates;  it  produces  acid  and  gas  in  media 
containing  dextrose,  levulose,  galactose,  lactose,  maltose,  and  man- 
nite.  It  develops  especially  well  on  media  containing  urine  or  bile. 

B.  coli  communis  does  not  peptonize  albumins;  it  does,  how- 
ever, break  down  some  of  the  higher  nitrogenous  compounds  into 
smaller  molecules.  Indol  is  one  of  the  most  important  products 
of  its  activity,  although  little  appears  to  be  formed  in  the  intestinal 
canal  in  health.  Nitrates  are  reduced  to  nitrites  and  from  them 
ammonia  and  free  nitrogen  are  produced. 

Resistance.  —  The  organisms  are  able  to  resist  a  higher  degree 
of  acidity  or  alkalinity  than  most  non-spore-bearing  forms.  They 
are  killed  in  from  five  to  ten  minutes  by  a  temperature  of  60°  C. 
Frozen  in  ice,  a  certain  percentage  will  live  for  six  months.  In 
carbolic  acid  1  to  100  they  are  destroyed  in  five  to  fifteen  minutes. 

Pathogenesis.  —  Intraperitoneal  injections  of  1  c.c.  or  more  of 
a  broth  culture  into  a  guinea  pig  or  rabbit  may  cause  death  within 


204  BACTERIOLOGY  FOR  NURSES 

twenty-four  to  forty-eight  hours.  There  is  a  rapid  decline  in 
temperature,  and  finally  the  development  of  a  fibrino-purulent 
peritonitis,  due  undoubtedly  to  the  endotoxins  liberated  from 
the  disintegrating  bacteria. 

In  man  they  are  considered  as  the  cause  of  the  majority  of 
cases  of  cystitis  and  should  such  an  infection  spread  they  may 
give  rise  to  pyelitis  or  suppurative  nephritis.  They  have  been 
isolated  from  abscesses  of  the  liver  and  gall-bladder.  Numerous 
epidemics  of  diarrhea  in  young  children,  cases  of  broncho-pneu- 
monia, pleurisy,  meningitis,  and  endocarditis,  have  also  been 
attributed  to  them.  In  ulcerative  conditions  of  the  intestines 
they  may  readily  pass  through  the  injured  intestinal  walls  and 
with  associated  organisms  give  rise  to  peritonitis.  Ordinarily  in 
such  cases  streptococci  and  staphylococci  are  also  present,  and  it 
is  probable  that  the  latter  are  more  actively  concerned  in  produc- 
ing the  lesions.  Shortly  before  death  the  colon  bacilli  frequently 
pass  through  the  intact  intestinal  mucosa  into  the  circulation. 

It  is  somewhat  surprising  that  an  organism  constantly  present 
in  such  large  numbers  in  the  intestines  should  at  times  give  rise 
to  disease;  it  might  naturally  be  expected  that  the  body  cells 
had  developed  a  complete  state  of  immunity  towards  it.  A 
number  of  explanatory  suggestions  have  been  offered.  It  may 
be  that  none  of  the  toxic  products  of  the  bacilli  are  absorbed 
through  the  intact  mucous  membranes,  in  which  case  no  process 
of  immunization  would  be  likely  to  occur ;  or,  on  the  other  hand, 
it  may  be  that  temporary  lowered  resistance  may  permit  the 
organisms  to  overcome  the  forces  by  which  they  have  previously 
been  held  in  check. 

Immunity.  —  Bacteriolytic  and  agglutinating  antibodies  are 
produced  in  animals  following  injections  of  gradually  increasing 
doses  of  living  or  dead  organisms.  The  normal  serum  of  animals 
and  man  will  frequently  agglutinate  B.  coli  in  dilutions  as  high  as 
1  in  10  or  1  in  20.  The  formation  of  such  agglutinins  may  probably 
be  the  result  of  their  habitual  presence  in  the  intestinal  tract. 
The  serum  of  patients  recovering  from  typhoid  fever  or  dysentery 
will  agglutinate  B.  coli  in  even  higher  dilutions.  The  fact  may  be 


PARATYPHOID  GROUP  205 

explained  either  on  the  ground  of  their  group  relationship  or  the 
absorption  of  the  toxic  substances  of  the  organism  through  the 
diseased  intestinal  mucous  membranes. 

Vaccines.  —  Vaccines  have  been  found  beneficial  in  cases  of 
cystitis  and  appendicitis  due  to  the  colon  bacillus.  The  dose 
ranges  from  25  to  500  million  organisms. 

B.  Coli  Communior.  —  The  organism  is  probably  as  abundant 
in  the  intestinal  tract  as  B.  coli  communis  itself.  Morphologically 
and  culturally  they  are  identical,  save  that  the  latter  does  not 
ferment  saccharose,  whereas  B.  coli  communior  is  able  to  form 
both  acid  and  gas  from  it. 

Capsulated  Bacilli.  —  Closely  related  to  the  colon  bacilli,  and 
by  some  authorities  included  with  them,  are  a  number  of  organ- 
isms which  differ  from  the  latter  in  that  they  are  non-motile  and 
that  they  are  usually  heavily  capsulated.  Of  these  B.  lactis 
aerogenes  is  perhaps  the  most  frequently  met  with.  It  is  normally 
present  in  the  intestines,  in  sewage,  and  in  water.  It  is  almost 
always  present  in  milk  and  cream  and  is  one  of  the  principal  agents 
causing  them  to  become  sour.  Another  of  the  capsulated  bacilli, 
B.  pneumonia  or  Friedlander's  pneumobacillus,  isolated  by  Fried- 
lander  in  1882,  was  regarded  for  a  time  as  the  cause  of  lobar 
pneumonia.  More  recent  discoveries  have  proved,  however, 
that  the  pneumococcus  is  responsible  for  the  vast  majority  of 
the  cases  of  the  disease.  The  type  of  pneumonia  caused  by 
Friedlander's  bacillus  is  relatively  infrequent  and  has  a  very 
high  mortality.  Still  other  members  of  the  capsulated  group  are 
B.  ozence  the  causal  agent  of  fetid  rhinitis,  and  B.  of  rhinoscleroma, 
which  receives  its  name  from  the  disease  it  gives  rise  to. 

PARATYPHOID   GROUP 

Between  the  colon  and  typhoid  bacilli  are  a  large  number 
of  organisms  which  possess  the  common  group  characteristics, 
i.e.  they  do  not  liquefy  gelatin,  are  Gram  negative,  and  do  not 
form  spores,  yet  they  show  wide  variations  in  their  reaction  on 
sugars  and  are  not  agglutinated  by  either  typhoid  or  coli  immune 
serum.  Near  the  typhoid  end  of  the  scale  organisms  exist  which 


206  BACTERIOLOGY  FOR  NURSES 

differ  culturally  from  the  typhoid  bacillus  in  that  they  produce 
acid  and  gas  from  glucose  while  the  former  produce  acid  only. 
At  the  other  end  of  the  scale  are  organisms  just  as  closely  related 
to  the  colon  bacilli.  Possibly  the  members  of  the  group  are 
links  in  a  chain  connecting  the  two  groups. 

Attention  has  centered  around  these  organisms  mainly  because 
of  their  connection  with  "  food  poisoning."  Such  poisoning  is 
usually  spoken  of  as  "  ptomain  poisoning,"  and  is  the  result  of 
poisonous  products  formed  from  the  food  itself.  The  food  at 
fault  may  not  appear  in  any  way  unusual,  a  bacteriological  ex- 
amination being  necessary  to  determine  the  presence  of  the 
bacteria.  Preserved  foods  or  sausages  are  the  most  frequent 
cause  of  poisoning ;  cases  have  been  reported  from  milk  and  milk 
products.  It  is  important  to  note  that  ptomains  are  relatively 
resistant  to  heat  and  that  the  organisms  themselves,  while  not 
especially  resistant,  may  escape  unharmed  by  the  usual  cooking 
processes  when  embedded  in  sausage  or  joints  of  meat. 

In  1888  in  a  village  in  Saxony  a  cow  which  had  been  sick  for 
two  days  with  profuse  diarrhea  was  slaughtered  and  the  meat 
sold  for  food.  Fifty-seven  persons  who  ate  the  meat  became  ill 
and  one  case  ended  fatally;  a  young  man  who  had  eaten  the 
meat  raw  died  in  about  thirty-six  hours.  From  this  fatal  case 
and  also  from  the  flesh  of  the  diseased  cow  Gartner  isolated  an 
organism  to  which  he  gave  the  name  B.  enteritidis.  As  its  name 
implies,  the  lesions  produced  by  the  organism  are  intestinal. 
Notably  an  inflammation  of  the  mucous  membrane  and  occasion- 
ally hemorrhages  occur.  When  infection  is  caused  by  the  bacillus 
itself  symptoms  do  not  usually  appear  until  about  twenty-four 
hours  or  more  after  the  food  has  been  eaten.  When  they  appear 
at  once  they  are  undoubtedly  due  to  the  poisonous  ptomains 
already  formed  from  the  food. 

Paratyphoid  Bacilli.  —  In  1896  Acharde  and  Bensaude  obtained 
from  the  urine  of  a  patient  suffering  from  an  infection  similar  to 
typhoid  fever  an  organism  which  they  named  the  paratyphoid 
bacillus.  In  1900-1901  Schottmiiller  isolated  from  the  blood  of 
patients  showing  much  the  same  symptoms  two  bacilli  closely 


PARATYPHOID   GROUP  207 

resembling  the  former,  which  he  termed  B.  paratyphoid  A  and 
B.  paratyphoid  B.  The  symptoms  produced  are  of  the  enteric 
form,  and  very  soon  the  organisms  appear  in  large  numbers  in  the 
stools  and  in  the  blood. 

Infection  with  Type  A  lasts  from  nine  to  fourteen  days  and  is 
characterized  by  headache,  pains  in  the  neck  and  back,  fever, 
and  occasionally  diarrhea.  Infection  with  Type  B  is  mani- 
fested by  a  more  sudden  onset  and  symptoms  very  similar  to 
those  produced  by  B.  enteritidis,  such  as  vomiting,  chills,  and 
diarrhea.  The  two  organisms  are  readily  distinguished  by  their 
production  of  specific  agglutinins. 

As  a  rule  paratyphoid  fever  is  much  milder  and  has  a  lower 
mortality  than  typhoid  fever.  It  is  not  known  what  degree  of 
immunity  is  conferred  by  one  attack,  but  it  is  known  that  an 
attack  does  not  protect  against  typhoid  nor  does  typhoid  protect 
against  paratyphoid.  When  exposure  to  both  infections  is  an- 
ticipated a  mixed  vaccine  is  usually  administered  in  doses  com- 
mencing with  500  millions  of  typhoid  bacilli  and  250  millions 
each  of  paratyphoid  A  and  B.  Second  and  third  doses  are  usually 
double  the  amount. 

Members  of  the  Group  Found  in  Animal  Diseases.  B.  suipes- 
tifer.  —  The  organism  was  isolated  from  cases  of  hog  cholera. 
It  is  usually  considered  as  a  secondary  invader ;  the  primary  cause 
of  the  disease  is  a  filtrable  virus.  The  organism  closely  resembles 
B.  paratyphoid  B,  and  can  only  be  distinguished  from  it  by  sero- 
logical  tests. 

B.  psittacosis.  —  Parrots  imported  from  the  tropics  often  die 
of  an  enteritis  and  general  septicemia  caused  by  this  organism. 
Rabbits,  pigeons,  fowls,  and  mice  are  also  susceptible.  From 
birds  or  animals  the  disease  is  readily  communicable  to  man  by 
the  infected  dejecta. 

Rat  Virus.  —  Dangez  isolated  an  organism  belonging  to  this 
group  from  an  epizootic  in  field  mice,  which  he  introduced  com- 
mercially for  the  purpose  of  killing  rats.  Other  similar  prepara- 
tions are  obtainable  in  the  market  under  the  names  of  Ratin, 
Liverpool  virus,  etc. 


CHAPTER  XX 

THE  COLON  TYPHOID  GROUP 

(CONTINUED) 

B.  TYPHOSUS.    DYSENTERY  GROUP 

B.  Typhosus.  —  In  1880  Eberth  found  the  organisms  now  known 
as  B.  typhosus  in  the  spleen  and  diseased  portions  of  the  intes- 
tines of  persons  who  had  died  of  typhoid  fever.  In  1884  Gaffky 
obtained  the  organism  in  pure  culture  and  was  able  to  study  its 
growth  characteristics.  Its  causal  relationship  to  typhoid  fever, 
however,  was  particularly  difficult  to  prove  because,  although 
the  organism  was  pathogenic  for  many  animals  when  inoculated 
subcutaneously  or  intravenously  it  was  impossible  to  produce 
infection  by  feeding  and  the  characteristic  symptoms  of  the  disease 
as  they  appear  in  man.  Experiments  with  anthropoid  apes, 
increased  knowledge  concerning  specific  antibodies  produced  in 
immune  serum,  the  presence  of  the  bacillus  in  the  blood  and  feces 
of  typhoid  patients  and  not  in  healthy  persons  other  than  "  car- 
riers "  have  been  sufficient  to  establish  the  fact  that  B.  typhosus 
is  the  causal  agent  of  typhoid  fever.  Recent  work  tends  to  show 
that  there  are  several  strains  slightly  different  culturally  which 
have  hitherto  been  classified  as  B.  typhosus,  and  that  in  the  future 
these  organisms  will  be  considered  as  the  typhoid  group  rather 
than  the  typhoid  bacillus. 

Morphology  and  Staining.  —  Typhoid  bacilli  are  short  rods 
with  rounded  ends,  varying  from  1  /*  to  3  /*  in  length  and  0.5 
to  0.8  7*  in  width.  In  hanging  drop  preparations  they  are  seen 
as  single  individuals  or  they  remain  attached  and  appear  as  threads. 
Morphologically  they  are  identical  with  B.  coli  save  that  they  are 

208 


BACILLUS   TYPHOSUS 


209 


FIG.  29.— Typhoid  Bacilli. 


often  more  slender  (Fig.  29).  When  growing  under  favorable 
conditions  they  are  actively  motile ;  in  hanging  drop  preparations 
made  from  young  cultures  the  short  forms  move  rapidly  with  a 
darting  movement,  while  the 
attached,  thread-like  forms 
have  a  slower  and  more  un- 
dulating motion.  They  pos- 
sess twelve  or  more  peritrichic 
flagella,  which  are  longer  and 
more  wavy  than  those  of  the 
colon  bacillus  (Fig.  30).  With 
the  ordinary  anilin  dyes  they 
stain  rather  slowly,  they  are 
Gram  negative,  and  do  not 
form  spores. 

Cultivation.  —  The  typhoid 
bacillus  is  aerobic  and  facultative  anaerobic.  Its  optimum  tem- 
perature is  about  37°  C.  On  culture  media  growth  does  not  take 
place  below  9°  C.  or  above  42°  C.  In  a  gelatin  stab  there  is  a 

fine  white  growth  along  the 
line  of  inoculation.  The  main 
growth,  however,  is  on  the  sur- 
face, which  spreads  outwards 
toward  the  sides  of  the  tube  as 
a  thin  leaf-like  film.  On  agar 
colonies  appear  in  twenty-four 
hours  as  thin  disks,  white  or 
bluish  gray  in  color,  and  with 
slightly  scalloped  margins. 
Broth  is  uniformly  clouded. 
Occasionally  a  thin  film  forms 
on  the  surface  after  eighteen 
to  twenty-four  hours'  growth. 
The  tests  with  sugars  furnish  a  means  of  differentiating  the 
typhoid  bacillus  from  other  members  of  the  colon-typhoid  group. 
The  typhoid  bacillus  produces  acid  without  gas  in  maltose,  glu- 


FIQ.  30.  —  Typhoid  Bacilli,  showing 
Flagella. 


210  BACTERIOLOGY  FOR  NURSES 

cose,  and  mannite,  but  has  no  effect  upon  lactose  or  saccharose. 
The  identification  of  the  organism  is  readily  established  by  means 
of  the  agglutination  reaction. 

Resistance.  —  Typhoid  bacilli  exhibit  about  the  same  degree 
of  resistance  to  heat  as  most  non-spore-bearing  organisms.  They 
are  killed  by  exposure  to  a  temperature  of  56°  C.  in  fifteen  minutes. 
Mercuric  chloride  1  to  1000  destroys  them  in  one  to  five  minutes 
and  carbolic  acid  1  to  100  in  five  to  fifteen  minutes.  As  a  rule 
they  are  less  resistant  to  disinfectants  than  B.  coli.  Such  substances, 
however,  as  brilliant  green  and  crystal  violet  inhibit  the  growth  of 
the  latter  without  affecting  the  typhoid  bacilli.  Application  has 
been  made  of  the  fact  in  preparing  special  media  for  the  isolation 
of  B.  typhosus  from  feces.  Since  the  organisms  are  rarely  found 
in  nature  it  is  difficult  to  determine  the  length  of  time  they  will 
live  outside  of  the  body.  In  feces  in  privy  vaults  or  on  the  ground 
they  tend  to  die  out  rapidly ;  the  majority  may  be  dead  within 
twenty-four  hours.  Some,  however,  may  persist  for  a  much  longer 
period.  According  to  certain  authorities  they  may  remain  alive 
in  feces  during  the  winter  for  five  months.  They  have  been  re- 
ported to  remain  alive  in  oysters  for  one  month ;  in  water  they 
seldom  live  longer  than  seven  days.  Laboratory  experiments 
show  that  in  ice  the  numbers  rapidly  decrease ;  a  certain  percentage 
may  live  for  four  months,  but  by  the  end  of  six  months  all  are 
killed. 

Pathogenesis.  —  There  is  no  evidence  that  the  typhoid  bacillus 
is  ever  associated  with  any  disease  in  animals  nor  is  any  disease 
known  amongst  animals  which  in  any  way  resembles  typhoid 
fever.  Subcutaneous  or  intraperitoneal  inoculations  of  pure 
cultures  produce  a  short,  acute  sickness  which  usually  ends  fatally 
in  from  twenty-four  to  forty-eight  hours,  but  which  in  no  way 
resembles  the  disease  as  it  occurs  in  man.  Attempts  to  produce 
the  disease  by  feeding  animals  on  typhoid  dejecta  mixed  with 
their  food  have  been  equally  unsuccessful  except  in  the  case  of 
anthropoid  apes. 

In  human  infection  inflammation  and  ulceration  in  the  Peyer's 
patches  and  solitary  glands  of  the  intestine  are  the  characteristic 


BACILLUS   TYPHOSUS  211 

lesions.  In  the  early  stage  of  the  disease  there  is  acute  inflamma- 
tion ;  leukocytes  gather  in  great  numbers  in  the  invaded  area  and 
suppuration  and  necrosis  result.  In  the  severest  cases  the  lesions 
may  involve  the  muscular  and  peritoneal  coats  of  the  intestinal 
wall  and  perforation  may  occur;  peritonitis  and  death  usually 
follow.  Passing  through  the  injured  mucous  membrane  into  the 
blood  stream  the  bacilli  are  carried  to  all  parts  of  the  body  and 
become  localized  in  groups  or  foci  in  the  various  organs.  Of  these 
the  spleen  usually  contains  the  greatest  number  of  bacteria;  it 
becomes  enlarged  and  congested  and  in  tissue  sections  the  bacilli 
appear  as  clumps  between  the  cells.  A  similar  but  less  marked 
invasion  may  take  place  in  the  liver  and  kidneys.  In  the  gall 
bladder  they  may  occur  in  enormous  numbers,  and  even  after  re- 
covery they  may  persist  there  for  years. 

In  addition  to  the  local  changes  in  the  various  organs  toxic  poi- 
soning is  manifested  in  typhoid  fever  as  in  other  infectious  diseases 
by  disturbances  in  the  circulatory,  respiratory,  and  heat  regulat- 
ing centers. 

Occasionally  complications  occur,  such  as  pneumonia,  osteo- 
myelitis, and  other  inflammatory  conditions,  in  which  the  typhoid 
bacillus  seems  to  be  the  exciting  cause.  Usually,  however,  such 
complications  are  due  to  a  secondary  or  mixed  infection  with 
staphylococci,  streptococci,  pneumococci,  or  the  colon  bacilli. 

The  bacilli  are  eliminated  mainly  in  the  feces.  About  the  second 
week  they  are  apt  to  appear  also  in  large  numbers  in  the  urine. 
Frequently  they  persist  until  several  weeks  or  months  after  con- 
valescence. Occasionally  they  are  found  in  the  secretions;  or- 
ganisms have  been  found  in  the  roseolar  spots  which  occur  in  ty- 
phoid. It  cannot  be  concluded,  however,  that  their  presence  is 
the  cause  of  such  spots.  In  cases  of  pneumonia  due  to  the  typhoid 
bacilli  they  are  abundantly  present  in  the  sputum.  There  is 
strong  evidence  that  the  bacilli  may  persist  in  the  gall  bladder 
for  many  years  and  from  thence  find  their  way  into  the  intestines. 
It  is  probable  that  the  catarrhal  inflammation  they  produce  there 
causes  a  deposit  of  the  bile  in  a  solid  form,  resulting  in  gallstones. 
In  operations  on  the  gall  bladder  years  after  recovery  from  the 


212  BACTERIOLOGY  FOR  NURSES 

disease  typhoid  bacilli  have  been  found.  They  have  even  been 
found  within  the  calculi. 

Typhoid  Carriers.  —  In  the  majority  of  cases  of  typhoid  fever 
the  bacilli  disappear  from  the  feces  during  the  first  three  or  four 
weeks  of  convalescence,  but  in  a  certain  number  of  cases,  about 
1  to  5  per  cent,  they  persist  for  many  months  and  even  years  after 
an  attack  of  the  disease.  Such  carriers  have  been  classified  as 
"  temporary  "  when  they  cease  excreting  the  bacilli  within  a  year 
of  convalescence  and  as  chronic  when  this  period  is  exceeded. 
The  distinction  is  unimportant  since  both  types  are  a  menace  to 
the  community  in  which  they  reside.  A  danger  lies  in  the  fact 
that  carriers  generally  appear  to  be  in  good  health  or  only  suffer 
occasionally  from  slight  pain  in  the  region  of  the  gall  bladder. 
The  majority  of  traced  carriers  are  women.  Since  in  such  cases 
the  chief  danger  lies  in  their  conveying  the  bacilli  to  foodstuffs, 
a  carrier  occupied  as  a  cook  or  waitress  or  on  a  dairy  farm  is  a 
special  menace.  The  following  remarkable  case  of  typhoid  carrier 
is  cited  by  Dr.  Park  of  New  York : 

"  A  visitor  in  the  family  of  which  this  woman  was  cook  developed 
typhoid  fever  some  ten  days  after  entering  the  household.  This 
was  in  1901.  The  cook  had  been  with  the  family  ten  years  and 
it  is  difficult  to  say  which  infected  the  other.  The  cook  went 
to  another  family.  One  month  later  the  laundress  in  this  family 
was  taken  ill. 

"  In  1902  the  cook  obtained  a  new  place.  Two  weeks  after  her 
arrival  the  laundress  was  taken  ill  with  typhoid  fever ;  in  a  week 
a  second  case  developed,  and  soon  seven  members  of  the  household 
were  sick. 

"  In  1904  the  cook  went  to  a  home  in  Long  Island.  There  were 
four  in  the  family  as  well  as  seven  servants;  within  three  weeks 
after  arrival  four  servants  were  attacked. 

"  In  1906  the  cook  went  to  another  family.  Between  August 
27  and  September  3  six  of  its  eleven  inmates  were  attacked  with 
typhoid.  At  this  time  the  cook  was  first  suspected.  She  entered 
another  family  on  September  21st.  On  October  5th  the  laundress 
developed  typhoid  fever. 


BACILLUS  TYPHOSUS  213 

"  In  1907  she  entered  a  family  in  New  York  City  and  two  months 
after  her  arrival  two  cases  developed,  one  of  which  proved  fatal. 

"  The  cook  was  removed  to  the  hospital  March  19,  1907.  Cul- 
tures taken  every  few  days  showed  bacilli  off  and  on  for  three 
years.  Sometimes  the  stools  contained  enormous  numbers  of 
bacilli  and  again  for  days  none  would  be  found.  She  was  released 
on  parole  in  1910,  promising  to  report  to  the  Health  Department 
and  not  to  engage  in  cooking.  She  broke  her  parole  and  disap- 
peared. In  1915  in  an  epidemic  of  typhoid  at  a  maternity  hos- 
pital a  total  of  twenty-five  cases  developed.  Investigation  showed 
that  the  food  was  the  cause  and  the  cook  was  identified  as  '  Ty- 
phoid Mary.'  During  the  period  of  disappearance  she  infected 
a  friend  and  was  the  cause  of  several  cases  in  a  small  private  sani- 
torium.  She  is  known  to  have  been  the  cause  of  at  least  fifty 
cases  of  typhoid  fever." 

The  tracing  of  typhoid  carriers  is  an  important  and  at  the  same 
time  difficult  problem.  A  Widal  reaction  cannot  be  depended 
upon  since  the  agglutinins  in  the  serum  vary  in  amount  from  time 
to  time.  The  actual  proof  that  a  person  is  a  carrier  lies  in  the 
isolation  of  the  typhoid  bacillus  from  the  feces  or  urine,  and  as  the 
organism  may  not  always  be  present,  several  examinations  must 
be  made  if  negative  results  are  at  first  obtained  in  suspected  cases. 

Modes  of  Communication.  —  The  typhoid  bacillus  probably 
always  enters  the  body  by  way  of  the  mouth,  fingers  or  food  being 
responsible  for  its  conveyance.  Water-borne  epidemics  still 
occur,  though  with  much  less  frequency.  Fortunately  typhoid 
bacilli  do  not  multiply  in  water;  they  usually  die  within  seven 
days  except  in  winter,  when  a  covering  of  ice  or  snow  affords 
them  some  protection.  Water-borne  epidemics  of  typhoid  almost 
always  occur  in  the  spring,  fall,  or  winter,  since  most  fecal  material 
eventually  finds  its  way  to  water,  and  as  watercourses  draining 
inhabited  regions  are  likely  to  be  contaminated  with  human  feces, 
there  is  always  the  possibility  of  surface  water  containing  typhoid 
bacilli.  The  first  big  epidemic  in  America  definitely  traced  to 
the  water  supply  occurred  in  1885  at  Plymouth,  a  small  mining 
town  near  Philadelphia.  Of  the  8000  inhabitants  1000  contracted 


214  BACTERIOLOGY  FOR  NURSES 

the  disease.  Plymouth  received  its  water  from  a  mountain  stream 
which  drained  an  almost  uninhabited  watershed.  The  infection 
was  traced  to  a  man  who  had  spent  his  Christmas  holidays  in 
Philadelphia,  had  contracted  the  disease  there,  and  had  returned 
home  in  January.  During  his  sickness  the  excreta  were  not  dis- 
infected but  were  thrown  oh  the  banks  or  into  the  frozen  stream. 
In  March  a  thaw  came  and  the  entire  mass  was  washed  into  the 
brook  and  on  into  the  water  main.  Three  weeks  later  the  disease 
began  to  appear  in  the  town  with  such  rapidity  that  some  days  as 
many  as  100  new  cases  were  reported.  In  all  there  were  114  deaths. 
The  epidemic  proved  at  least  that  freezing  alone  for  a  short  period 
is  not  sufficient  to  destroy  the  organism. 

Milk-borne  epidemics,  like  those  due  to  water,  have  a  sudden 
onset  and  then  subside  rather  sharply.  Up  to  1907  statistics 
showed  317  epidemics  caused  by  infected  milk;  Most  milk  out- 
breaks are  reported  from  England  or  America.  The  custom  of 
boiling  the  milk  in  many  other  countries  undoubtedly  affords  them 
a  certain  amount  of  protection  against  typhoid  infection.  Milk- 
borne  epidemics  usually  have  certain  definite  characteristics.  As 
a  rule  contamination  comes  from  a  case  or  a  carrier  on  the  farm 
and  the  outbreak  is  localized  to  the  area  receiving  milk  from 
that  farm.  Usually  people  of  the  better  class  and  those  who 
drink  milk  raw  are  affected,  and  several  cases  may  occur  simul- 
taneously in  one  house.  Milk  products  have  been  responsible 
for  a  certain  number  of  outbreaks;  oysters  and  other  shellfish 
have  also  contributed  their  quota.  Vegetables  such  as  celery, 
lettuce,  and  water  cress  grown  on  land  fertilized  with  fresh  night 
soil  may  account  for  a  few  cases. 

Flies  have  been  justly  condemned  as  spreaders  of  the  disease. 
They  breed  in  fecal  and  decomposing  masses  of  all  kinds  and  de- 
posit the  organisms  they  accumulate  on  the  food  they  walk  over. 
In  a  recent  experiment  typhoid  bacilli  were  isolated  from  five  out 
of  eighteen  flies  captured  in  the  privy  and  on  a  fence  near  the  sick 
room  of  a  typhoid  patient. 

A  number  of  cases  occur  due  to  lack  of  knowledge  of  caring 
for  the  sick.  The  danger  of  fomites  containing  living  bacilli 


BACILLUS   TYPHOSUS  215 

is  very  real,  and  the  utmost  care  in  disinfecting  all  articles  used 
by  a  patient  as  well  as  all  excreta  cannot  be  overemphasized. 

Immunity.  —  One  attack  of  typhoid  fever  usually  confers  im- 
munity which  lasts  for  several  years ;  in  about  2  per  cent  of  persons 
having  had  one  attack  a  second  attack  occurs  which  is  usually 
very  mild.  Immune  serum  is  highly  bactericidal  and  possesses 
abundant  agglutinins,  precipitins,  and  opsonins.  Gradually  in- 
creasing doses  of  living  or  dead  bacilli  injected  into  animals  pro- 
duces a  similar  serum,  but  attempts  to  use  it  therapeutically  have 
not  met  with  much  success. 

Serum  Diagnosis.  —  The  fact  that  the  serum  of  typhoid  patients 
will  in  high  dilutions  agglutinate  typhoid  bacilli,  while  the  serum 
of  normal  individuals  or  those  not  suffering  from  the  disease  has 
no  effect  upon  them,  has  been  of  enormous  aid  in  the  diagnosis  of 
typhoid  fever.  The  first  application  of  the  test  was  reported  by 
Widal  in  1896.  Details  of  the  method  have  been  given  in  a  previous 
chapter.  Usually  the  reaction  is  given  about  the  seventh  day 
and  gradually  increases  until  convalescence.  In  about  95  per  cent 
of  all  cases  it  is  said  to  appear  at  some  period  of  the  disease. 

Bacterial  Diagnosis.  —  A  blood  culture  is  generally  positive 
during  the  first  week  of  the  disease  in  all  cases ;  during  the  second 
week  in  about  50  per  cent  of  cases,  and  as  the  disease  progresses 
the  organisms  tend  to  disappear  from  the  blood  stream. 

Bacilli  seem  to  be  most  numerous  in  the  feces  during  the  second, 
third,  and  fourth  weeks  of  the  disease.  The  short  life  of  the  or- 
ganisms outside  of  the  body  makes  it  imperative  that  specimens 
be  examined  as  soon  after  passage  as  possible. 

A  small  portion  of  the  feces  is  emulsified  if  solid  in  a  tube  of 
broth,  if  fluid  it  can  be  plated  without  further  preparation.  Poured 
plates  are  made  of  special  media  such  as  that  of  Conradi-Dri- 
galsky,  and  a  loopful  of  the  fecal  material  is  streaked  over  the 
solidified,  media.  The  usual  method  is  to  use  three  plates  for 
each  specimen,  streaking  the  second  and  third  plates  without 
recharging  the  loop.  After  twenty-four  hours'  incubation  a  blue 
typhoid-like  colony  is  fished  into  broth  and  at  the  end  of  from 
eight  to  ten  hours  sufficient  growth  will  have  developed  for  the 


216  BACTERIOLOGY  FOR  NURSES 

next  procedure.  Hanging  drop  preparations  are  made  of  a  mix- 
ture of  the  broth  culture  and  a  dilution  of  immune  horse  serum, 
the  strength  of  which  is  already  known.  With  appropriate  dilu- 
tions agglutination  will  take  place  almost  immediately  if  the  or- 
ganism tested  is  the  typhoid  bacillus.  The  result  may  be  confirmed 
by  inoculating  media  containing  lactose  and  dextrose.  Acid  pro- 
duction will  take  place  in  the  latter  and  no  change  in  the  former. 

Vaccines.  —  Wonderfully  good  results  have  been  obtained  from 
the  injection  of  killed  bacilli  as  a  prophylactic  measure  against 
typhoid  fever  both  in  military  and  civil  life.  Statistics  show  a 
steady-  decline  of  typhoid  in  the  U.  S.  Army  since  the  introduc- 
tion of  compulsory  vaccination  in  1910.  Only  one  case  occurred 
in  1913  among  over  80,000  men.  In  the  British  Army  the  reduc- 
tion of  morbidity  is  estimated  at  50  per  cent.  An  excessive  dose 
of  infectious  material  may  break  down  the  protection  resulting 
from  the  action  of  the  vaccine,  yet  in  such  cases  the  severity  of  the 
disease  will  be  considerably  modified. 

In  the  army  500  million,  1  billion,  and  10  billion  bacteria  are 
given  usually  on  three  successive  Saturdays  by  means  of  a  sub- 
cutaneous injection  near  the  insertion  of  the  deltoid  muscle.  Oc- 
casionally a  slight  local  inflammation  and  a  general  feeling  of 
malaise  develops  which  disappears  within  twenty-four  to  forty- 
eight  hours.  Consequently  it  is  customary  to  give  the  vaccine  in 
the  afternoon  so  that  any  reaction  which  may  develop  will  occur 
while  the  individual  is  in  bed.  The  degree  of  immunity  decreases 
after  two  and  a  half  years.  It  is  advisable,  however,  in  cases  of 
constant  strain  and  exposure  to  revaccinate  each  year. 

Attempts  have  been  made  to  use  small  doses  of  vaccine  as  a 
therapeutic  measure  in  typhoid  fever.  Excellent  results  are 
reported  in  a  certain  number  of  cases;  in  others,  however,  they 
have  been  unsatisfactory. 

THE  DYSENTERY  GROUP 

The  term  dysentery  is  usually  applied  to  diseases  which  show 
such  symptoms  as  intestinal  pain  and  diarrhea  with  mucus  and 


THE  DYSENTERY  GROUP  217 

blood  in  the  stools.  Within  recent  times  two  distinct  forms  have 
been  distinguished :  one  variety,  amebic  dysentery,  is  caused  by  a 
protozoon ;  the  other  form,  badllary  dysentery,  is  caused  by  bacilli 
of  the  colon-typhoid  group. 

In  1898  Shiga,  a  Japanese  bacteriologist,  isolated  an  organism 
from  the  stools  of  dysentery  patients  which  he  found  would  agglu- 
tinate with  the  serum  of  those  patients  and  not  with  that  of  normal 
individuals.  Moreover,  he  was  not  able  to  find  the  organism  in  the 
feces  of  patients  suffering  from  other  diseases  nor  in  those  of  normal 
individuals.  Shiga's  bacillus  is  now  considered  the  causal  agent 
of  the  majority  of  acute  dysentery  epidemics  which  occur  in  tem- 
perate climates. 

In  1899  Flexner,  while  investigating  dysentery  in  Manila,  isolated 
a  bacillus  from  dysenteric  stools  which  at  that  time  he  considered 
identical  with  that  isolated  by  Shiga,  but  later  found  that  it  dif- 
fered in  agglutinative  reactions.  In  the  same  year  Kruse  in  Ger- 
many isolated  similar  bacilli  from  cases  of  dysentery.  In  1902 
Park  and  Dunham  obtained  an  organism  from  a  severe  case  of 
dysentery  at  Seal  Harbor,  Mt.  Desert,  Maine,  which  proved  by 
its  different  agglutinating  characteristics  to  be  still  another  strain. 
Since  that  time  several  others  have  been  described.  One  writer 
has  even  described  fifteen  different  forms  which  have  fermentative 
characteristics  distinguishing  them  one  from  the  other.  The 
following  classification  of  Hiss,  in  which  all  members  fall  into  one 
of  four  groups,  is  the  generally  accepted  one : 

Type  1.     Shiga.     Ferments  dextrose. 
Type  2.     Park-Hiss.     Ferments  dextrose  and  mannite. 
Type  3.     Flexner-Strong.     Ferments  dextrose,  mannite,  and  saccharose. 
Type  4.     Harris-Wollstein.     Ferments  dextrose,  mannite,  saccharose,  and 
maltose. 

Of  the  four  types  it  is  generally  agreed  that  Type  1  appears  most 
frequently  in  the  severest  forms  of  the  disease ;  types  3  and  4  are 
found  more  frequently  than  the  others  in  the  dysentery  or  summer 
diarrhea  of  young  children. 

Morphology  and  Staining.  —  The  organisms  closely  resemble 
the  typhoid  bacilli  save  that  they  are  somewhat  thicker,  and 


218  BACTERIOLOGY  FOR  NURSES 

filamentous  forms  rarely  are  seen.  Their  staining  reactions 
are  the  same  as  those  of  other  members  of  the  colon-typhoid 
group. 

Cultivation.  —  In  gelatin  stab  cultures  a  thin  line  of  growth 
develops,  very  little  appearing  on  the  surface.  Colonies  on  agar 
and  gelatin  plates  are  much  the  same  as  those  of  the  typhoid  bacilli 
and  are  smaller  and  more  transparent  than  those  of  B.  coli.  In 
broth  a  uniform  cloudiness  is  produced  with  sometimes  a  pellicle 
or  a  slight  deposit.  As  already  stated  the  different  strains  behave 
differently  towards  the  different  sugars ;  they  all  ferment  dextrose 
and  none  of  them  are  able  to  ferment  lactose. 

Resistance.  —  Dysentery  bacilli  show  much  the  same  degree 
of  resistance  to  heat  and  disinfectants  as  the  typhoid  bacilli.  In 
feces  they  usually  die  in  one  or  two  days. 

Pathogenesis.  —  With  the  exception  of  monkeys  the  charac- 
teristic disease  cannot  be  produced  in  animals  by  feeding  them 
with  cultures  of  the  bacilli.  Many  animals,  however,  are  sensi- 
tive to  subcutaneous  or  intravenous  inoculations,  and  the  surpris- 
ing result  of  such  inoculation  is  that  the  animals  show  all  the  symp- 
toms of  the  disease,  and  on  autopsy  the  mucous  membrane  of  the 
cecum  and  colon  are  found  to  be  excessively  inflamed.  It  is  evident 
that  the  cells  of  the  intestinal  mucous  membrane  have  a  strong 
affinity  for  the  bacterial  toxin. 

In  man  the  organism  does  not  enter  the  blood  stream  and  the 
lesions  are  especially  confined  to  the  intestinal  mucous  membrane. 
In  mild  cases  the  disease  takes  the  form  of  a  catarrhal  inflamma- 
tion only ;  in  severer  cases  necrosis  of  the  epithelium  may  occur 
and  the  intestines  may  be  lined  with  a  pseudomembrane  consisting 
of  fibrin,  dead  cells,  and  bacteria. 

Since  the  bacilli  are  found  only  in  the  intestines  the  spread  of 
the  disease  is  due  to  fecal  contamination  direct  or  indirect.  Food, 
soiled  linen,  carriers :  all  may  play  a  part.  Water  may  become 
contaminated  as  in  the  case  of  typhoid,  although  comparatively 
few  water-borne  epidemics  of  dysentery  have  been  reported.  It  is 
stated  that  in  Japan  in  the  rural  districts  the  mortality  due  to 
dysentery,  resulting  mainly  from  the  use  of  human  feces  as  a  fer- 


THE  DYSENTERY  GROUP 


219 


tilizer  and  the  frequent  infection  of  the  small  streams  and  wells, 
is  over  20  per  cent. 

Immunity.  —  Bacteriolysins  and  agglutinins  are  abundantly 
produced  in  the  immune  serum  of  both  human  beings  and  animals. 
Since  there  are  so  many  strains  of  dysentery  bacilli  it  is  customary 

FERMENTATION   REACTIONS   OF   THE    PRINCIPAL    MEMBERS 
OF  THE  COLON-TYPHOID   GROUP 


ORGANISM 

m 
Q 

LEVULOSE 

MANNITE 

MALTOSE 

LACTOSE 

SACCHAROSE 

g 

s 
I 

1 

c 

B    coli  communis  m 

44 

44 

44 

44 

44 



4 

4 

C 

t 

B.  coli  communior     .     .     .     .      m 

44 

44 

44 

44 

44 

44 

4 

4 

C 

B    acidi  lactici       — 

_l__l_ 

44 

44 

44 

44 



4- 

4 

C 

1 

B.  mucosus  capsulatus  ...     — 

+  + 

+  + 

+  + 

+  + 

+  + 

+  + 

+ 

+ 

B    enteritidis                   .     .     .      in 

44 

44 

_l_. 

44 

B.  paratyphosus  A    ....      m 

4-4 

— 

— 

— 

— 

B.  paratyphosus  B     .     .     .     .      m 

+  + 

+  + 

+  + 

+  + 

— 

— 

— 

— 

B    typhosus      m 

4 

4 

4 

4 

B.  dysenteric,  Shiga     ...     — 









B.  dysenteric,  Park       ...     — 

4 

+ 

+ 

— 

— 

— 

— 

— 

B.  dysenteric,  Flexner  ...     — 

+ 

4 

4 

4 

— 

— 

— 

— 

B.  dysenteric,  Wollstein-Harris  — 

+ 

+ 

+ 

+ 

+ 

m  =  motility 
4-  +  =  acid  and  gas  production 


+  =acid  production  only 
C  =  coagulation 


to  inject  animals  with  an  individual  strain  and  so  produce  a  "  mon- 
ovalent  "  serum  or  to  inject  them  with  mixed  strains  and  so  give 
rise  to  a  "  polyvalent "  serum,  the  former  for  therapeutic  use  when 
the  type  of  organism  causing  the  disease  has  been  determined  and 
the  latter  when  the  strain  is  unknown.  In  mild  cases  doses  from 
10  c.c.  to  30  c.c.  are  given  twice  daily  according  to  the  weight  of 
the  patient ;  in  severe  cases  as  much  as  100  c.c.  may  be  given. 


220  BACTERIOLOGY  FOR  NURSES 

The  reduction  in  mortality  by  the  use  of  the  serum  is  estimated  at 
about  20  per  cent. 

Vaccines.  —  Dysentery  vaccines  have  been  employed  with  mod- 
erately good  results.  The  fact  that  there  are  so  many  strains 
lessens  their  value  unless  it  is  known  to  which  group  the  invading 
organisms  belong. 

Bacteriological  Diagnosis.  —  Isolation  from  the  feces  is  the  sur- 
est method  of  identifying  the  strain  causing  infection.  The  meth- 
ods employed  are  exactly  the  same  as  those  employed  in  typhoid 
and  paratyphoid  infection  except  that  crystal  violet  should  be 
omitted  from  the  Conradi  medium  on  account  of  the  inhibitory 
effect  of  the  anilin  dyes  on  many  of  the  dysentery  strains.  After 
isolation  the  organism  should  be  tested  with  the  specific  agglu- 
tinating serum  and  confirmatory  evidence  gained  by  growing  it 
on  the  different  sugar  media. 


'    CHAPTER  XXI 

BACILLUS  ANTHRACIS.     BACILLUS  MALLEI.     BACIL- 
LUS  PYOCYANEUS.     BACILLUS   PROTEUS. 

B.  Anthracis.  —  Anthrax  or  splenic  fever  is  a  disease  occurring 
especially  in  sheep  and  cattle,  although  many  of  the  lower  animals 
are  susceptible.  Infection  occasionally  appears  in  human  beings, 
but  it  is  never  transmitted  from  man  to  man ;  it  is  always  con- 
tracted directly  or  indirectly  from  animals. 

Anthrax  has  undoubtedly  occurred  among  cattle  from  the 
earliest  times.  It  was  the  first  infectious  disease  shown  to  be 
caused  by  a  specific  microorganism  and  consequently  it  has  been 
one  of  the  most  studied  of  all  bacterial  diseases.  Pollender  in 
1849  described  rod-shaped  bodies  which  were  contained  in  the 
blood  of  infected  animals  and  suggested  that  they  might  be  the 
cause  of  the  disease.  In  1863  Davaine  demonstrated  by  inocula- 
tion experiments  that  blood  containing  these  bodies  invariably 
produced  anthrax.  He  therefore  concluded  they  were  bacteria 
and  suggested  the  name  B.  anthracis.  Later,  in  1877,  Koch 
confirmed  Davaine's  work  by  isolating  the  organism,  growing 
it  in  pure  culture,  and  with  the  pure  culture  producing  the  char- 
acteristic disease.  Koch's  observations  explained  many  apparent 
paradoxes  that  had  greatly  puzzled  previous  workers.  Blood 
from  infected  animals  which  appeared  to  be  free  from  bacteria 
had  been  found  to  produce  the  specific  disease  when  inoculated 
into  susceptible  animals  and  also  the  disease  was  sometimes 
found  to  appear  without  any  known  means  of  infection.  Koch 
discovered  that  very  soon  after  blood  is  drawn  the  anthrax  bacillus 
forms  spores  which  are  highly  refractive  and  seen  with  difficulty, 
and  that  consequently  they  must  have  been  present  but  not 

221 


222 


BACTERIOLOGY  FOR  NURSES 


noticed  in  the  supposedly  germ-free  blood.  Further  research 
showed  that  animals  might  become  infected  by  feeding  them  with 
spores.  This  fact  together  with  a  knowledge  of  the  prolonged 
vitality  of  these  bodies  in  the  soil  explained  the  persistence  of  the 
disease  in  certain  localities  and  its  reappearance  in  once-infected 
pastures  after  many  years. 

Morphology  and  Staining.  —  The  bacillus  is  one  of  the  largest 
of  the  pathogenic  bacteria;  it  ranges  from  2  to  20  ft  in  length 

and  1  to  1.2  /*  in  width.  In 
stained  film  preparations  the 
organisms  may  appear  singly 
or  joined  end  to  end  in  chains 
of  varying  length.  The  free 
ends  of  the  rods  are  rounded, 
while  those  coming  in  contact 
with  one  another  are  square  or 
slightly  swollen  and  concave, 
the  latter  giving  the  chain 
somewhat  the  appearance  of  a 
bamboo  rod.  In  preparations 
from  albuminous  material  a 
thin  capsule  may  be  seen  surrounding  the  cell.  (Fig.  31.) 

Spores  are  formed  only  in  the  presence  of  free  oxygen ;  hence 
they  do  not  develop  in  the  blood  while  it  remains  in  the  body. 
They  are  oval  in  shape  and  appear  in  the  center  of  the  rod.  As 
the  spore  develops  it  occupies  more  and  more  of  the  parent  cell 
until  the  latter  appears  as  a  thin  envelope  which  finally  ruptures 
and  sets  the  spore  free.  Sporeless  strains  of  B.  anthracis  have  been 
produced  by  growing  the  organisms  on  media  containing  anti- 
septics or  by  cultivation  at  their  maximum  temperature  (43°  C.). 
The  bacilli  stain  with  the  usual  dyes  and  are  Gram  positive. 

Cultivation.  —  The  organism  grows  well  on  ordinary  culture 
media  under  aerobic  conditions ;  vegetative  forms  are  facultative 
anaerobes.  Development  will  occur  between  14°  C.  and  43°  C., 
the  optimum  being  about  34°  C. ;  under  the  minimum  and  above 
the  maximum  temperature  sporulation  does  not  take  place. 


FIG.  31.  —  Anthrax  Bacilli. 


BACILLUS  ANTHRACIS  223 

In  gelatin  stab  cultures  growth  occurs  along  the  track  of  the 
needle  as  a  delicate  white  thread  from  which  irregular  projections 
soon  extend,  giving  the  culture  the  appearance  of  an  inverted  tree ; 
at  the  end  of  two  or  three  days  liquefaction  commences  at  the  top. 
In  broth  after  twenty-four  hours'  incubation  growth  appears  as 
a  flaky  sediment  which  is  deposited  at  the  bottom  of  the  tube. 
The  colonies  on  agar  or  gelatin  plates  are  particularly  character- 
istic. At  first  they  appear  as  small,  white,  opaque  points ;  later 
long,  wavy  filaments  project  from  each  colony  in  all  directions, 
which  when  examined  through  the  microscope  are  seen  to  be 
composed  of  bacteria  joined  end  to  end. 

Resistance.  —  Anthrax  spores  retain  their  vitality  and  virulence 
for  years  under  favorable  conditions.  Exposed  to  dry  heat,  a 
temperature  of  140°  C.  for  three  hours  is  required  to  kill  them; 
moist  heat  has  a  much  more  rapid  effect,  a  temperature  of  100°  C. 
being  sufficient  to  destroy  them  in  five  minutes.  They  have  been 
found  to  retain  their  vitality  after  thirty-six  days'  exposure  to  a 
5  per  cent  solution  of  carbolic  acid  at  room  temperature.  In  a 
similar  solution,  however,  they  were  destroyed  after  half  an  hour's 
exposure  at  55°  C.  In  the  vegetative  form  B.  anthracis  has  com- 
paratively low  resistance. 

Pathogenesis.  —  Cattle  and  sheep,  except  the  Algerian  race, 
are  the  most  frequently  infected  of  all  animals.  In  European 
countries  an  outbreak  is  apt  to  occur  from  time  to  time ;  in  France 
the  animal  mortality  among  sheep  was  formerly  about  10  per  cent. 
An  animal  may  suddenly  show  symptoms  of  collapse  and  death 
ensue  within  a  few  minutes ;  or  in  milder  cases,  bloody  mucus  is 
seen  about  the  mouth  and  nose  and  in  the  feces,  pulse  and  respira- 
tion are  increased,  and  chills  are  followed  by  high  temperature. 
In  such  cases  death  may  occur  in  from  twelve  to  forty-eight  hours. 
In  still  less  severe  cases  edema  and  often  ulceration  and  necrosis 
of  the  neck  lymph  glands  occur.  On  autopsy  the  spleen  is  found 
to  be  soft  and  of  a  dark  red  color  and  two  or  three  times  its  natural 
size.  Tissue  sections  show  the  capillaries  both  of  the  spleen  and 
liver  packed  with  bacilli ;  the  blood  is  usually  fluid  but  tar-like, 
and  of  a  dark  color. 


224  BACTERIOLOGY  FOR  NURSES 

In  man  one  of  three  forms  of  infection  may  occur :  entrance  of 
the  bacilli  may  be  through  a  cut  or  an  abrasion  of  the  skin,  result- 
ing in  a  malignant  'pustule;  through  the  lungs  by  inhalation  of  the 
spores,  wool  sorter's  disease;  or  through  the  alimentary  tract,  in- 
testinal anthrax. 

When  infection  takes  place  through  the  skin  a  small  red  papule 
appears  on  the  exposed  surface  in  about  one  to  three  days.  Very 
soon  it  becomes  vesicular  and  contains  clear  or  blood-stained 
fluid.  The  area  surrounding  it  becomes  greatly  inflamed  and 
within  thirty-six  hours  the  center  begins  to  show  signs  of  necrosis. 
If  the  pustule  is  not  excised  the  disease  spreads.  Invasion  of  the 
blood  stream  by  the  bacilli  is  most  likely  to  happen,  and  death 
from  septicemia  result  in  from  three  to  five  days.  Occasionally 
instead  of  the  typical  pustule  an  extensive  edematous  area  appears 
which  may  be  so  intense  as  to  result  in  gangrene.  Such  cases  are 
usually  fatal.  Skin  infections  develop  chiefly  among  shepherds 
and  butchers  or  those  who  work  among  hides. 

The  pulmonic  form  of  anthrax,  "  wool  sorter's  disease,"  is 
contracted  by  the  inhalation  of  spores  during  the  sorting  and 
cleansing  of  wool  from  infected  animals.  The  symptoms  are 
those  of  pneumonia,  often  with  edema  in  the  cutaneous  tissue 
over  the  neck  and  chest.  Recovery  may  occur  or  the  disease 
may  be  fatal  in  from  two  to  seven  days. 

Intestinal  anthrax,  although  the  usual  form  in  cattle,  rarely 
occurs  in  man.  The  few  instances  on  record  have  been  caused  by 
the  ingestion  of  spore-infected  food  or  accidentally  amongst 
laboratory  workers.  The  symptoms  produced  are  those  of  in- 
tense poisoning,  chills,  vomiting  and  diarrhea,  and  a  moderate 
degree  of  fever. 

Immunity.  —  With  the  hope  of  producing  protective  immunity 
Pasteur,  in  1880-1882,  devised  a  method  whereby  a  mild  attack 
of  the  disease  could  be  produced  by  means  of  inoculation  with 
attenuated  cultures.  Other  methods  have  since  been  suggested, 
but  that  employed  by  Pasteur,  Chamberland,  and  Roux  is  the 
one  still  most  generally  used. 

Two  vaccines  are  prepared  :  No.  1  is  a  broth  culture  so  attenu- 


BACILLUS   MALLEI  225 

ated  by  cultivation  at  42°  to  43°  C.  that  it  no  longer  affects  guinea 
pigs  but  is  still  fatal  for  mice ;  No.  2  is  sufficiently  virulent  to  kill 
guinea  pigs  but  not  rabbits.  The  animal  to  be  immunized  is 
inoculated  subcutaneously  on  the  inner  side  of  the  thigh  with 
five  drops  of  vaccine  No.  1 ;  twelve  days  later  a  similar  dose  of 
vaccine  No.  2  is  injected ;  fourteen  days  later  virulent  organisms 
can  be  injected  without  any  ill  results.  It  is  estimated  that  in 
about  40  per  cent  of  all  the  animals  vaccinated  immunity  dis- 
appears within  a  year  and  that  to  insure  permanent  protection 
revaccination  would  be  necessary  every  year.  Nevertheless,  the 
system  has  done  much  to  diminish  the  mortality  from  the  disease. 
In  France  statistics  show  that  during  twelve  years  the  mortality 
from  anthrax  amongst  vaccinated  sheep  was  less  than  1  per  cent 
as  compared  with  10  per  cent  in  flocks  not  thus  protected. 

A  moderately  protective  serum  has  been  obtained  from  actively 
immunized  animals,  but  the  nature  of  its  protection  is  not  definitely 
known ;  it  is  thought  to  be  largely  due  to  the  presence  of  opsonins. 
A  combination  of  the  active  and  passive  methods  of  immunization 
has  been  employed  with  the  result  that  a  single  treatment  is  said  to 
suffice. 

B.  Sub  tills.  —  In  the  early  days  of  bacteriology  B.  subtilis,  a 
saprophytic  organism  found  widely  distributed  in  nature,  was 
thought  to  be  closely  related  to  B.  anthracis  because  of  their 
almost  identical  morphological  appearance.  B.  subtilis,  com- 
monly known  as  the  "  hay  "  bacillus,  may  be  distinguished,  how- 
ever, by  its  motility  and  by  its  non-pathogenicity. 


BACILLUS   MALLEI 

Glanders  is  an  infectious  disease  primarily  of  horses,  mules, 
and  asses ;  occasionally  it  is  transmitted  to  other  animals  and  to 
man.  Towards  the  end  of  1882  Loeffler  and  Schutz  demonstrated 
conclusively  the  causal  relationship  of  B.  mallei  to  the  disease 
by  isolating  the  organism  from  diseased  tissues  and  experimentally 
producing  the  characteristic  symptoms  by  inoculating  it  into 
animals. 
Q 


226  BACTERIOLOGY  FOR  NURSES 

Morphology  and  Staining.  —  The  bacillus  is  a  small  rod  straight 
or  slightly  curved  with  rounded  or  pointed  ends,  ranging  from  1.5 
to  5  /A  in  length  and  0.25  to  0.5  /*  in  width;  it  usually  occurs 
singly  but  may  occasionally  be  seen  in  pairs  or  long  filaments. 
The  organism  is  non-motile  and  does  not  form  spores ;  it  does  not 
stain  readily  with  the  ordinary  aniline  dyes  and  is  Gram  negative. 

Cultivation.  —  Growth  occurs  on  ordinary  culture  media  be- 
tween 22°  and  43°  C. ;  a  slightly  acid  reaction  to  phenolphthalein 
and  the  addition  of  glycerin  greatly  favors  development.  Stroke 
cultures  on  agar  or  glycerin  agar  at  37°  C.  are  somewhat  trans- 
parent and  of  a  grayish  white  color  and  a  rather  slimy  consistency. 
On  agar  plates  colonies  appear  as  round  transparent  droplets; 
in  broth  a  diffuse  cloudiness  appears  which  later  collects  at  the 
bottom  of  the  tube  as  a  heavy  viscous  sediment.  Growth  on 
potato  is  particularly  characteristic ;  about  the  third  day  a  yellow- 
ish transparent  layer,  somewhat  like  clear  honey,  is  visible;  as 
growth  continues  the  color  deepens  until  by  the  seventh  or  eighth 
day  it  is  of  a  chocolate-brown  color  while  the  surrounding  potato 
has  acquired  a  greenish  yellow  tint. 

Resistance.  —  B.  mallei  like  other  vegetative  forms  is  only 
feebly  resistant  to  heat  and  antiseptics.  It  is  killed  by  exposure 
to  moist  heat  at  55°  C.  in  ten  minutes  and  in  a  5  per  cent  solution 
of  carbolic  acid  in  from  three  to  five  minutes.  It  is  somewhat 
resistant  to  drying.  It  has  been  found  to  retain  its  vitality  for 
fourteen  days  in  a  dry  condition. 

Pathogenesis.  —  As  already  stated,  glanders  occurs  chiefly 
among  horses;  sheep,  goats,  swine,  and  rabbits  are  relatively 
less  susceptible,  cattle  are  immune. 

The  disease  occurs  in  man  as  a  result  of  the  direct  contact  of 
a  wound  or  skin  abrasion  with  the  discharges  or  diseased  tissues 
of  an  infected  animal.  Consequently  only  those  who  come  directly 
in  contact  with  horses  are  likely  to  be  affected.  In  animals  the 
lesions  are  of  two  types,  usually  spoken  of  as  "  glanders  "  and 
"  farcy."  In  glanders  proper  the  nasal  mucous  membranes 
become  inflamed  and  a  profuse  catarrhal  discharge  appears. 
Very  soon  firm  translucent  nodules  are  formed  which  later  soften 


BACILLUS  MALLEI  227 

in  the  center,  break  down,  and  leave  irregular  ulcerated  cavities. 
Similar  lesions  occur  in  the  lungs,  in  the  liver,  and  in  the  spleen. 
In  "  farcy  "  the  infection  usually  takes  place  through  an  abrasion 
of  the  skin;  the  lymphatics  near  the  wound  become  thickened 
and  tense  and  are  spoken  of  as  "  farcy  pipes  "  or  "  farcy  buds  " ; 
Suppuration  usually  follows,  resulting  in  deep  ulcers  with  ragged 
edges  and  frequently  a  purulent  discharge. 

In  man  the  disease  occurs  either  in  an  acute  or  a  chronic  form. 
In  the  acute  form  an  inflammatory  swelling  appears  at  the  point 
of  infection,  which  is  usually  the  hand  or  arm,  and  a  redness 
spreads  along  the  line  of  the  lymphatics  as  in  a  poisoned  wound. 
A  pustular  eruption  soon  appears  which  may  be  local  or  cover  a 
large  area ;  in  addition  suppurative  foci  may  occur  in  the  lungs 
and  other  internal  organs.  In  about  60  per  cent  of  all  cases  the 
disease  ends  fatally  in  from  two  to  three  weeks. 

In  the  chronic  form  a  local  ulcer  forms  which  tends  to  spread 
deeply  and  superficially;  the  disease  may  run  a  chronic  course 
for  years  and  recovery  may  eventually  occur,  or,  on  the  other 
hand,  it  may  at  any  time  change  into  the  acute  form  and  rapidly 
become  fatal. 

The  glanders  nodule  has  been  considered  by  some  authorities 
to  be  structurally  similar  to  that  formed  by  the  tubercle  bacillus. 
It  is  generally  agreed,  however,  that  in  glanders  there  is  a  more 
marked  inflammatory  reaction  and  leukocytic  infiltration  and 
that  the  tissue  changes  are  of  a  degenerative  rather  than  of  a 
proliferative  nature.  Caseation,  which  is  so  marked  in  tubercu- 
losis, does  not  occur  in  the  same  degree  in  glanders,  nor  are  the 
typical  giant  cells  formed. 

The  mode  of  infection  amongst  horses  is  not  definitely  known. 
Recent  evidence  tends  to  show  that  it  takes  place  mainly  by  the 
way  of  the  alimentary  tract.  Since  the  bacilli  are  numerous  in 
the  nasal  discharge,  the  public  drinking  trough  may  in  a  measure 
be  responsible  for  the  spread  of  the  disease.  In  man  infection 
probably  only  occurs  through  a  break  in  the  skin. 

Diagnosis.  —  Several  methods  have  been  devised  which  greatly 
facilitate  the  diagnosis  of  glanders ;  of  these  the  mallein  reaction, 


228  BACTERIOLOGY  FOR  NURSES 

the  Straus  reaction,  and  sero-diagnostic  tests  are  the  most  fre- 
quently employed. 

Mallein  Reaction.  —  Mallein  is  a  concentrated  glycerin  broth 
in  which  B.  mallei  has  been  cultivated  and  is  prepared  in  exactly 
the  same  manner  as  tuberculin.  After  a  subcutaneous  injection 
a  positive  reaction  in  a  glandered  animal  is  manifested  by  a  rise 
in  temperature  of  about  two  degrees,  a  tender  local  swelling  at  the 
point  of  inoculation,  and  a  general  disturbance,  while  in  healthy 
horses  the  temperature  does  not  rise  above  one  degree  and  the 
local  swelling  is  slight  and  soon  disappears. 

It  has  been  recently  shown  that  mallein  dropped  into  the  con- 
junctival  sac  gives  a  similar  reaction  to  the  ophthalmic  test  in 
tuberculosis.  Three  drops  of  mallein  dropped  into  the  eye  of  a 
glandered  animal  will  cause  a  swelling  of  the  eyelid  and  a  purulent 
discharge  from  the  tested  eye  in  from  five  to  six  hours.  The  test 
is  so  simple  and  gives  such  a  reliable  and  quick  result  that  it  has 
been  adopted  as  the  Federal  test  for  the  interstate  shipment  of 
horses. 

The  Straus  reaction  consists  in  injecting  into  the  peritoneal 
cavity  of  a  male  guinea  pig  some  of  the  suspected  material.  If 
virulent  glanders  bacilli  are  present,  enlargement  of  the  testicles 
and  pus  formation  occurs  within  two  to  five  days.  A  positive 
reaction  together  with  the  presence  of  typical  organisms  in  the 
lesion  is  proof  positive  of  the  disease.  Failure  on  the  part  of  the 
guinea  pig  to  react,  however,  does  not  preclude  the  possibility  of 
the  disease. 

Serum  Reactions.  —  The  serum  of  an  infected  horse  possesses 
a  very  high  power  of  agglutination ;  a  dilution  of  1  to  1000  or  higher 
will  react  positively.  Since  normal  serum  will  agglutinate  the 
bacilli  in  dilutions  as  high  as  1  to  500,  three  dilutions  of  serum 
from  a  suspected  animal  are  generally  employed :  1  to  500,  1  to 
800,  and  1  to  1000.  If  agglutination  occurs  only  with  the  first 
dilution  the  reaction  is  considered  negative;  with  the  first  and 
second,  doubtful ;  with  the  first,  second,  and  third,  positive. 

Complement  fixation  tests  give  probably  the  most  reliable 
results  of  all.  According  to  certain  workers  a  positive  reaction  is 


BACILLUS   PYOCYANEUS  229 

obtained  in  about  97  per  cent  of  all  positive  cases  of  the  disease. 
The  test  is  conducted  as  described  in  Chapter  XIII,  save  that 
several  strains  of  B.  mallei  are  used  together  as  an  antigen. 

All  attempts  to  produce  artificial  immunity  against  glanders 
have  so  far  been  unsuccessful. 


BACILLUS  PYOCYANEUS 

The  blue  green  color  occasionally  seen  in  the  purulent  dis- 
charge of  wounds  of  long  standing  was  shown  by  Gessard,  in  1882, 
to  be  due  to  a  chromogenic  bacillus  to  which  was  given  the  name 
B.  pyocyaneus. 

Morphology  and  Staining.  —  The  organism  is  a  slender  rod 
from  2  to  6  /i  long  and  0.3  to  1  p  broad ;  it  possesses  a  single 
flagellum  at  one  end  and  is  actively  motile.  It  stains  with  the 
ordinary  aniline  dyes,  is  Gram  negative,  and  does  not  form  spores. 

Cultivation.  —  The  bacillus  is  an  aerobeand  facultative  anaerobe. 
It  grows  readily  on  all  artificial  culture  media  and  gives  to  most  of 
them  a  bright  green  color.  On  gelatin  growth  rapidly  develops, 
imparting  to  the  medium  the  characteristic  hue;  liquefaction 
commences  about  twenty-four  hours  after  inoculation  and  soon 
the  entire  medium  becomes  fluid.  On  agar  a  wrinkled  yellowish 
white  surface  growth  appears,  the  agar  itself  being  a  brilliant 
green.  In  broth  a  heavy  pellicle  is  formed  and  indol  is  produced ; 
milk  becomes  curdled  and  shows  an  alkaline  reaction. 

The  bacillus  produces  two  pigments :  one  a  fluorescent  green 
which  is  soluble  in  water  but  not  in  chloroform  and  which  is 
common  to  many  bacteria ;  and  another,  pyocyanin,  which  is  of 
a  blue  color  and  soluble  in  chloroform.  Pigment  production  is 
most  abundant  in  the  presence  of  oxygen  and  at  a  temperature 
of  about  22°  C. 

In  addition  to  the  ferment  causing  the  liquefaction  of  gelatin 
another  enzyme,  pyocyanose,  is  produced,  which  acts  on  albumin 
and  is  able  to  dissolve  bacteria.  It  has  been  applied  locally  in 
cases  of  diphtheria.  The  results,  however,  have  not  been  markedly 
beneficial. 


230  BACTERIOLOGY  FOR  NURSES 

Pathogenesis.  —  B.  pyocyaneus  has  been  found  in  water,  in 
the  feces  of  many  animals,  and  on  the  skin  of  healthy  human 
beings,  and  for  some  time  after  its  isolation  it  was  regarded  as  a 
harmless  saprophyte  or  at  most  of  very  limited  pathogenic  power. 
Later  evidence  has  proved  that  not  only  does  its  presence  in 
mixed  infections  retard  the  process  of  repair,  but  it  has  the  power 
of  producing  suppuration  itself.  It  has  been  found  in  pure  cul- 
tures in  cases  of  ophthalmia,  broncho-pneumonia,  and  otitis  media. 
Thus  while  only  slightly  pathogenic  it  may  in  cases  of  lowered 
vitality  produce  a  serious  infection. 

BACILLUS  PROTEUS 

B.  proteus,  or  rather  the  group  of  organisms  known  by  that 
name,  is  abundantly  found  in  soil  and  water  and  almost  wherever 
putrefactive  changes  in  organic  matter  are  occurring.  The  or- 
ganisms were  discovered  by  Hauser  in  1885.  The  number  of  va- 
rieties contained  in  the  group  has  not  been  clearly  defined.  The 
following  description,  however,  may  be  considered  as  typical. 

Morphology  and  Staining.  —  The  average  length  is  about  1.2 
p  and  the  width  about  0.6  p.  The  organism  does  not  form 
spores.  It  possesses  many  peritrichal  flagella,  is  actively  motile, 
stains  readily  with  the  anilin  dyes,  and  is  Gram  negative. 

Cultivation.  —  An  aerobe  and  facultative  anaerobe,  it  grows  well 
on  the  ordinary  culture  media.  Gelatin  liquefaction  commences 
at  the  end  of  ten  or  twelve  hours.  On  agar  slants  a  spreading, 
white,  moist  growth  appears  and  on  agar  plates  colonies  tend  to 
become  confluent.  Dextrose  and  saccharose  are  fermented  with 
the  production  of  acid  and  gas.  In  peptone  solution  indol  and 
phenol  are  produced.  In  urine  the  organisms  decompose  urea 
into  ammonium  carbonate. 

Pathogenesis.  —  Cultures  injected  subcutaneously  into  guinea 
pigs  or  rabbits  cause  purulent  abscesses  or  death  with  symptoms 
of  poisoning. 

In  man  B.  proteus  has  been  found  in  a  variety  of  pathological 
conditions:  in  purulent  peritonitis,  cystitis,  and  pyelonephritis. 


BACILLUS   PROTEUS  231 

Metchnikoff  regarded  it  as  the  usual  cause  of  infantile  diarrhea. 
On  nasal  membranes  it  frequently  occurs  as  a  more  or  less  harm- 
less parasite,  decomposing  the  secretions  and  giving  rise  to  a 
putrefactive  odor.  Certain  outbreaks  of  food  poisoning  have 
been  attributed  to  the  "  ptomains "  produced  by  the  putre- 
factive action  of  B.  proteus.  In  such  cases  the  food  is  disagree- 
able both  in  taste  and  odor.  Hence  food  poisoning  of  this  type  is 
less  likely  to  occur  than  that  due  to  the  paratyphoid-enteritidis 
group  or  to  B.  botulinus,  where  there  is  little  or  no  perceptible 
change. 


CHAPTER  XXII 

(1)  HEMOGLOBINOPHILIC  GROUP.  (2)  HEMORRHAGIC 
SEPTICEMIA  GROUP 

(1)  B.  INFLUENZA.     B.  OF  KOCH-WEEKS.     B.  PERTUSSIS. 
B.  OF  SOFT  CHANCRE 

B.  Influenzas.  —  Influenza  was  described  as  early  as  the  fifteenth 
century  although  in  all  probability  it  was  known  even  earlier. 
It  has  appeared  in  all  parts  of  the  world  sporadically  in  epidemics 
or  in  great  pandemics.  In  1889-1890  so  widespread  was  the  disease 
and  so  high  the  mortality  that  it  was  considered  the  most  serious 
pandemic  of  modern  times.  During  the  two  years  following 
many  attempts  were  made  to  discover  the  specific  cause,  and  in 
January,  1892,  Pfeiffer,  Kitasato,  and  Canon  simultaneously  pub- 
lished a  description  of  the  organism  now  known  as  B.  influenzas. 
Pfeiffer's  work  was  the  most  complete  and  to  it  is  due  most  of 
the  knowledge  we  possess  of  the  organism.  B.  influenzas  has 
been  definitely  shown  to  be  pathogenic  and  is  generally  accepted 
as  the  cause  of  the  disease  although  the  fact  has  not  been  abso- 
lutely proved. 

Morphology  and  Staining.  —  The  organism  is  one  of  the  smallest 
pathogenic  bacteria  known.  As  seen  in  film  preparations  from 
sputum  it  averages  from  0.5  to  1.5  p  in  length  and  0.2  to  0.3  /*> 
in  width.  The  ends  of  the  rod  are  rounded  and  no  capsule  is 
formed.  The  organism  is  Gram  negative  and  is  colored  rather 
faintly  with  the  ordinary  anilin  dyes.  Staining  is  best  effected 
with  a  1  in  10  solution  of  carbol  fuchsin  for  five  to  ten  minutes. 
It  is  non-motile  and  does  not  form  spores. 

Cultivation.  —  Pfeiffer  succeeded  in  growing  the  organism  in 
symbiosis  with  others  on  agar  smeared  with  sputum,  but  all  at- 

232 


BACILLUS   INFLUENZA    ft  233 

tempts  to  cultivate  it  alone  on  plain  agar  or  serum  utterly  failed. 
He  then  tried  smearing  the  agar  with  drops  of  blood,  and  his  efforts 
were  completely  rewarded.  The  necessary  substance  for  their 
development  seems  to  be  hemoglobin,  and  for  this  reason  the  or- 
ganism is  spoken  of  as  "  hemoglobinophilic."  On  blood  agar 
colonies  appear  at  the  end  of  eighteen  hours  as  minute  circular 
almost  transparent  dots.  Even  on  blood  culture  medium  the 
organisms  very  soon  die.  They  will  live  indefinitely,  however,  if 
transplanted  every  three  or  four  days.  Grown  in  symbiosis  with 
other  organisms  development  is  more  rapid,  and  growth  will  occur 
for  several  generations  on  ordinary  agar  without  the  addition  of 
hemoglobin.  The  organism  is  a  strict  ae'robe  and  multiplies 
only  at  a  temperature  between  25°  C.  and  42°  C. 

Resistance.  —  The  bacillus  is  extremely  delicate  and  has  only 
very  feeble  powers  of  resistance.  In  dried  sputum  it  dies  within 
twelve  to  forty-eight  hours ;  in  water  it  does  not  live  more  than 
two  days ;  it  cannot  withstand  boiling  for  one  minute  or  a  tem- 
perature of  60°  C.  for  five  minutes. 

Pathogenesis.  —  B.  influenzse  is  only  slightly  virulent  for  ex- 
perimental animals ;  the  rabbit  is  moderately  susceptible  and  the 
guinea  pig  even  less.  There  is  no  satisfactory  evidence  that  they 
ever  contract  the  disease  in  a  natural  way. 

In  man  the  organisms  appear  in  enormous  numbers  in  the 
secretions  of  the  nose,  throat,  and  respiratory  tract.  Frequently 
they  invade  the  lung  tissue,  and  lobular  pneumonia,  purulent 
in  character,  results.  The  bronchioles  become  filled  with  leukocytes 
and  in  tissue  sections  the  bacilli  may  be  seen  packed  in  between 
the  epithelial  and  pus  cells.  The  bacilli  are  rarely  found  in  the 
blood.  They  may,  however,  be  present  in  the  lesions  accompanying 
influenza.  They  have  been  found  in  inflammations  of  the  middle 
ear,  in  meningitis,  conjunctivitis,  cystitis,  and  peritonitis. 

Influenza  may  take  a  subacute  form.  Bacilli  may  remain  latent 
or  only  slightly  virulent  in  the  lung  tissue  for  many  months,  and 
then  if  by  chance  the  body  resistance  is  lowered  they  may  become 
active. 

Infection  is  undoubtedly  transmitted  directly  from  one  indi- 


234  BACTERIOLOGY  FOR  NURSES 

vidual  to  another  or  indirectly  by  the  use  of  objects  contami- 
nated with  fresh  secretions  since  the  organism  is  so  feebly  resistant 
to  influences  outside  of  the  body,  and  it  is  present  in  enormous 
numbers  in  the  secretions  of  the  respiratory  tract.  Carriers  may 
be  responsible  for  the  cases  which  appear  sporadically  and  for  the 
commencement  of  epidemics. 

Immunity.  —  No  apparent  immunity  seems  to  be  conferred  by 
an  attack  of  the  disease ;  in  fact,  one  attack  seems  to  predispose 
to  subsequent  attacks.  Nor  has  a  serum  been  produced  which 
could  be  used  for  the  production  of  passive  immunity;  vaccine 
treatment  has  been  said  to  have  been  of  benefit.  Its  value,  how- 
ever, has  not  yet  been  fully  confirmed. 

KOCH-WEEKS  BACILLUS 

The  organism  was  first  observed  by  Koch  in  1883  while  in  Egypt, 
and  later  in  1887  it  was  more  fully  described  by  Weeks  in  New 
York,  who  obtained  it  in  cultures  growing  with  B.  xerosis  from  cases 
of  "  pink  eye  "  or  acute  contagious  conjunctivitis.  The  bacillus 
is  closely  similar  to  the  influenza  bacillus ;  the  relationship  has  not 
yet,  however,  been  determined.  Recent  studies  indicate  that 
the  Koch- Weeks  bacillus  may  be  a  strain  of  the  influenza  bacillus. 

Several  similar  hemoglobinophilic  organisms  have  been  de- 
scribed in  pathological  conditions  of  the  eye.  In  1896  Morax  and 
later  Axenfeld  isolated  the  Morax-Axenfeld  bacillus  from  cases 
of  subacute  inflammation  of  the  eye.  Zur  Nedden  found  a  similar 
bacillus  known  by  his  name  in  certain  ulcers  of  the  cornea. 

BACILLUS  PERTUSSIS 

Bordet  and  Gengou  in  1906  were  the  first  to  describe  an  organism 
resembling  the  influenza  bacillus  which  appears  in  enormous 
numbers  in  the  sputum  of  cases  of  whooping  cough  and  to  which 
they  gave  the  name  B.  pertussis. 

Morphology  and  Staining.  —  The  bacillus  is  a  short,  somewhat 
oval  rod,  usually  appearing  singly,  but  occasionally  in  pairs  joined 


BACILLUS   PERTUSSIS  235 

end  to  end;  it  is  non-motile.  It  stains  faintly  with  the  aniline 
dyes.  Bi-polar  bodies  which  stain  more  deeply  than  the  center  of 
the  organism  can  sometimes  be  distinguished.  It  is  decolorized  by 
Gram's  method. 

Cultivation.  —  The  organism  grows  feebly  at  first  even  on  the 
medium  especially  recommended  by  Bordet  and  Gengou,  consist- 
ing of  1  per  cent  glycerin  mixed  with  macerated  potato  and  an 
equal  quantity  of  human  or  rabbit  blood.  After  several  genera- 
tions it  will  grow  moderately  well  on  veal  agar  or  broth.  The 
organism  is  an  aerobe  and  develops  best  at  37°  C. 

Pathogenesis.  —  A  mild  toxin  is  undoubtedly  produced  and 
absorbed,  but  of  more  importance  is  the  mechanical  disturbance 
caused  by  the  bacilli  in  the  respiratory  tract.  They  have  been 
described  as  being  present  in  enormous  numbers  in  the  trachea, 
packed  between  and  clinging  to  the  cilia  of  the  epithelial  cells. 
This  according  to  certain  investigators  constitutes  the  specific 
lesion  of  whooping  cough.  B.  pertussis  has  not  yet  been  definitely 
proved  to  be  the  specific  cause  of  the  disease,  although  much  evi- 
dence has  been  advanced  in  favor  of  the  theory.  Its  strongest 
claim  to  recognition  is  the  fact  that  in  the  serum  of  convalescents 
a  specific  antibody  is  produced  which  gives  a  positive  complement 
fixation  reaction  with  the  organism. 

BACILLUS   OF  SOFT   CHANCRE 

The  bacillus  of  soft  chancre  or  chancroid  was  obtained  in  pure 
culture  by  Ducrey  in  1889  from  the  purulent  discharge  of  an 
ulcerated  surface. 

Morphology  and  Staining.  —  The  organism  is  exceedingly  small, 
measuring  about  1.5  /^  in  length  and  0.5  ^  in  width.  In  tissue 
sections  it  usually  appears  attached  in  long  chains  or  grouped 
together  in  masses.  It  is  non-motile  and  does  not  form  spores 
and  is  Gram  negative.  Stained  with  carbol  fuchsin  deeply  colored 
bi-polar  bodies  can  often  be  distinguished. 

Cultivation.  —  The  best  medium  for  cultivation  has  been  found 
to  be  a  mixture  of  agar  and  rabbit's  blood  in  the  proportion  of 


236  BACTERIOLOGY  FOR  NURSES 

two  parts  of  agar  to  one  part  of  blood.  In  fact,  on  no  other  culture 
medium  so  far  employed  has  cultivation  been  possible.  If  pus 
obtained  from  a  lesion  is  smeared  over  the  medium  minute  gray 
transparent  colonies  will  usually  appear  after  forty-eight  hours 
incubation. 

Pathogenesis.  —  The  disease  appears  as  an  acute  inflammation 
followed  in  one  or  two  days  by  pustule  formation.  The  pustule 
soon  ruptures  and  a  small  depressed  ulcer  remains,  around  which 
other  pustules  and  ulcers  develop  and  necrosis  spreads  rapidly. 
The  lymphatics  in  the  groin  become  swollen,  and  later  "  buboes  " 
or  abscesses  result.  The  lesions  are  usually  upon  the  genitals 
and  infection  is  most  frequently  conveyed  from  one  person  to 
another  by  direct  contact.  The  chancre  differs  from  that  of 
syphilis  in  that  there  is  no  induration. 

So  far  animal  inoculations  have  been  without  result  except  in 
monkeys.  The  causal  relationship  of  the  organism  to  the  disease, 
however,  was  established  by  Tomasczewski,  who  produced  the 
lesions  by  the  injection  of  a  pure  culture  into  a  human  body. 

(2)  HEMORRHAGIC   SEPTICEMIA   GROUP 

A  group  of  bacilli  have  been  described  all  of  which  have  similar 
characteristics  and  which  give  rise  in  the  lower  animals  to  an  acute 
septicemia  usually  with  hemorrhagic  areas  in  the  subcutaneous 
tissues  and  internal  organs.  The  bacilli  are  short,  non-motile, 
Gram  negative,  and  they  do  not  form  spores;  growth  is  scanty 
on  gelatin  and  the  medium  is  not  liquefied.  .  They  have  been 
found  in  rabbit  septicemia,  chicken  cholera,  swine  plague,  and  a 
similar  infection  in  cattle. 

Morphologically  and  culturally  the  organisms  isolated  from  the 
different  sources  appear  identical.  They  vary,  however,  in  their 
degree  of  virulence  for  the  different  animal  species.  B.  Avisepticus 
produces  a  septicemia  rapidly  fatal  in  fowls  but  much  less  severe 
in  pigs,  sheep,  and  horses.  B.  Suisepticus  is  moderately  pathogenic 
for  fowls,  but  in  young  pigs  it  produces  a  broncho-pneumonia,  to 
which  they  usually  quickly  succumb.  Evidence  points  to  a  close 


BACILLUS   PESTIS 


237 


relationship  between  the  members  of  the  group.  So  far  as  is  known 
none  of  the  organisms  affecting  the  lower  animals  are  pathogenic 
for  man  except  a  very  similar  organism,  B.  Pestis,  which  is  respon- 
sible for  the  much-dreaded  bubonic  plague  or  "  black  death." 

B.  Pestis.  —  Records  of  the  ravages  of  the  bubonic  plague  have 
been  handed  down  through  the  centuries.  At  intervals  it  has 
appeared  in  vast  epidemics  and  so  great  has  been  the  infectiousness 
and  mortality  of  the  disease  that  in  congested  districts  whole 
populations  have  succumbed.  The  "  Great  Plague "  of  the 
fourteenth  century  spread  over  all  Europe,  and  so  frightful  was 
its  severity  that  one  quarter  of  the  population  or  about  25,000,000 
persons  perished.  Commerce 
was  suspended  and  people  fled 
panic  stricken  from  the  towns 
to  the  open  fields  for  safety. 
During  the  last  two  centuries 
Western  Europe  has  been  prac- 
tically free  from  the  disease. 
It  still  occurs,  however,  in  all 
its  horror  in  India,  the  annual 
mortality  averaging  about 
500,000.  It  is  thought  that 
the  disease  made  its  first  ap- 
pearance in  America  in  1899  at 
Santos,  Brazil ;  since  then  other  cases  have  been  reported  in  San 
Francisco,  Mexico,  and  Central  America. 

The  causal  agent  of  the  disease,  B.  pestis,  was  discovered  simul- 
taneously by  Kitasato  and  Yersin  in  1894  during  an  epidemic  in 
China.  A  number  of  accidental  infections  with  pure  cultures 
have  established  its  specificity. 

Morphology  and  Staining.  —  In  film  preparations  from  infected 
tissues  the  bacilli  appear  as  short,  thick  rods  with  rounded  ends 
about  1.6  /*  in  length  and  0.6  p  in  width.  In  body  fluids  they 
may  be  seen  singly  or  in  pairs  and  rarely  in  chains ;  in  broth  cul- 
tures, on  the  contrary,  they  remain  attached,  and  the  individual 
organisms  are  so  short  and  thick  as  to  give  almost  the  appearance 


FIG.  32.  —  Bacillus  Pestis. 


238  BACTERIOLOGY  FOR  NURSES 

of  streptococci.  Many  varieties  in  form  are  seen  in  smears  of 
material  from  old  lesions.  Swollen  involution  forms  with  clubbed 
ends,  short,  round  forms,  and  long  rods  may  all  be  found,  most  of 
which  stain  with  difficulty.  (Fig.  32.) 

Young  forms  stain  well  with  methylene  blue,  which  brings  out 
clearly  the  deeper  stained  bi-polar  bodies;  they  are  decolorized 
by  Gram's  method.  The  organism  is  non-motile  and  does  not 
form  spores. 

Cultivation.  —  The  optimum  temperature  for  B.  pestis  is  some- 
what lower  than  that  of  most  pathogenic  organisms ;  growth  occurs 
better  at  25°  C.  to  30°  C.  than  at  37°  C.  The  bacillus  is  an  aerobe 
and  grows  well  on  all  ordinary  media  which  have  a  slightly  alkaline 
reaction.  On  nutrient  agar  or  gelatin,  colonies  present  a  delicate 
transparent  appearance  which  when  seen  under  the  low-power 
lens  show  a  granular  center  with  a  thin,  uneven  margin.  The  most 
characteristic  growth  takes  place  in  broth,  where,  if  the  tubes  are 
not  disturbed,  a  pellicle  forms  on  the  surface,  and  from  it  long,  deli- 
cate filaments  are  suspended  which  hang  down  into  the  broth  like 
stalactites  from  the  roof  of  a  cave.  Gelatin  is  not  liquefied;  a 
small  amount  of  acid  is  produced  from  dextrose,  but  no  other  sugars 
are  fermented. 

Resistance.  —  B.  pestis  exhibits  little  resistance  to  heat,  dry- 
ing, and  disinfectants.  In  cultures  protected  from  light  and  air 
they  have  been  found  alive  after  ten  years. 

Pathogenesis.  —  Plague  is  primarily  a  disease  of  rodents  trans- 
missible to  man.  In  all  probability  these  animals  are  responsible 
for  the  maintenance  of  foci  from  which  the  great  epidemics  spring. 
Of  the  lower  animals  rats,  mice,  guinea  pigs,  and  squirrels  are 
particularly  susceptible ;  dogs,  swine,  cattle,  and  horses  do  not 
contract  the  disease  naturally,  but  may  be  infected  by  inoculation 
with  large  amounts  of  cultures.  Mice  and  guinea  pigs  are  usually 
employed  for  experimental  purposes.  After  inoculation  a  local 
inflammatory  swelling  appears  which  follows  the  line  of  the  lym- 
phatics and  terminates  in  a  general  infection  and  death  in  a  few 
days.  On  autopsy  the  internal  organs  present  a  congested  appear- 
ance accompanied  by  extensive  hemorrhages.  The  liver  and  spleen 


BACILLUS   PESTIS  239 

are  enlarged  and  show  a  characteristic  granular  or  mottled  appear- 
ance, sometimes  with  abscess  formation  and  necrosis.  Rats  and 
mice  may  also  be  infected  by  feeding  them  with  pure  cultures  or 
the  dead  carcasses  of  their  infected  comrades.  In  such  cases  it 
is  thought  that  infection  takes  place  through  the  mucous  mem- 
branes of  the  mouth  rather  than  by  the  intestinal  canal.  The 
fact  that  infection  may  take  place  through  slight  abrasions  of  the 
skin  serves  a  useful  purpose  in  diagnosis.  Often  rats  submitted 
for  examination  are  decidedly  decomposed.  If,  however,  the  sus- 
pected material  is  rubbed  on  to  the  freshly  shaved  abdomen  of  a 
guinea  pig  the  plague  bacilli  enter  through  the  slight  scarification 
due  to  shaving  and  give  rise  to  a  general  infection,  whereas  the  other 
organisms  present  are  not  able  to  penetrate  the  skin. 

In  man  three  clinical  types  of  plague  are  recognized  :  (1)  bubonic, 
(2)  pneumonic,  and  (3)  septicemic.  In  the  bubonic  form  the 
lymphatic  glands  become  intensely  inflamed  and  swollen,  ending 
in  necrotic  softening  if  the  patient  lives  long  enough .  The  surround- 
ing connective  tissue  is  similarly  affected,  and  often  subcutaneous 
hemorrhages  occur,  causing  the  dark  colored  spots  which  originated 
in  the  middle  ages  the  popular  name  of  "  black  death."  Usually 
one  group  of  glands  is  first  affected,  and  from  this  primary  "  bubo  " 
the  swelling  and  necrosis  extends  to  other  groups.  Hemorrhage 
and  necrosis  may  also  occur  in  the  lungs,  liver,  and  spleen. 

In  the  pulmonary  form  the  disease  appears  as  a  broncho-pneu- 
monia often  attended  by  hemorrhages;  there  is  usually  a  large 
amount  of  frothy,  blood-tinted  sputum  in  which  the  bacilli  are 
present  in  enormous  numbers.  In  this  form  the  disease  is  ex- 
tremely infective  and  almost  always  fatal.  The  mortality  is  esti- 
mated to  be  90  per  cent  or  more. 

In  plague  septicemia  there  is  a  slight  general  enlargement  of 
the  lymphatic  glands,  but  no  primary  bubo  is  discoverable.  A  case 
commencing  in  the  bubonic  form  may,  however,  terminate  with 
septicemia.  In  the  bubonic  and  the  septicemic  types  the  bacillus 
remains  in  the  diseased  organs  and  the  blood  stream  and  is  not 
eliminated  in  the  excretions.  These  forms  of  the  disease  therefore 
are  not  contagious  in  the  usual  sense  of  the  word,  but  are  spread 


240  BACTERIOLOGY  FOR  NURSES 

mainly  through  the  agency  of  the  flea.  On  the  other  hand,  the 
pneumonic  form  is  frequently  transmitted  from  one  person  to 
another  by  careless  disposal  of  the  sputum  or  droplet  infection. 
The  mode  by  which  the  bacilli  enter  the  body  does  not  necessarily 
determine  the  type  of  infection  which  will  ensue. 

The  above  three  types  of  the  disease  are  usually  classified  as 
pestis  major;  milder  forms  occur  known  as  pestis  minor,  which  bear 
somewhat  the  same  relationship  to  the  severer  form  that  "  walk- 
ing typhoid  "  does  to  typhoid  fever. 

Modes  of  Infection.  —  When  first  it  was  noticed  that  an  epi- 
demic of  plague  was  accompanied  by  an  increased  mortality 
amongst  rats  a  supposition  at  once  arose  that  there  must  be  some 
relationship  between  rat  plague  and  human  plague,  and  the  possible 
mode  of  the  conveyance  of  the  disease  from  rat  to  man  or  from  rat 
to  other  rodent  became  a  question  of  prime  interest.  As  a  result  of 
experiments  it  soon  became  evident  that  rat  fleas  were  responsible 
for  the  transfer  of  the  infection.  It  was  noticed  that  infected 
animals  might  be  placed  in  a  cage  with  healthy  animals  without 
the  latter  contracting  the  disease  if  fleas  were  excluded;  on  the 
other  hand,  healthy  animals  placed  near  enough  to  flea-infested 
plague  rats  invariably  contracted  the  disease.  It  was  also  found 
that  the  common  rat  flea  infests  and  bites  human  beings;  large 
numbers  were  found  on  the  legs  of  men  who  entered  for  a  short 
time  plague-infected  houses.  The  ease  with  which  bacilli  may 
enter  the  tissues  was  also  demonstrated  by  allowing  a  non-infected 
flea  to  bite  a  rat  and  then  placing  a  drop  of  culture  over  the  bite 
after  which  infection  resulted.  Since  the  blood  of  an  infected 
rat  may  contain  as  many  as  100,000,000  bacilli  per  c.c.  an  enormous 
number  may  be  present  even  in  the  amount  of  blood  sucked  by  a 
flea.  The  organisms  multiply  further  in  the  stomach  of  the  in- 
sect, which  may  remain  infected  for  one  or  two  months.  Trans- 
ference of  the  organism  to  man  probably  takes  place  by  means  of 
the  feces  expelled  by  the  insect  while  feeding  or  the  regurgitation 
of  infected  blood  previously  taken  into  the  stomach.  Once  de- 
posited on  the  skin  it  is  an  apparently  simple  matter  for  the  bacilli 
to  enter  the  skin  through  the  minute  opening  made  by  the  flea 


BACILLUS   PESTIS  241 

and  infect  the  surrounding  tissues.  The  pneumonic  types  of 
plague  may  dev.elop  after  infection  by  a  flea  if  the  organisms  enter- 
ing the  blood  stream  happen  to  become  localized  in  the  lungs. 

Immunity.  —  One  attack  of  plague  as  a  rule  confers  immunity. 
A  method  of  protective  inoculation  with  "  Haffkine's  prophylac- 
tic "  is  extensively  practiced  in  India  which  has  given  very  good 
results.  The  "  prophylactic  "  is  prepared  by  inoculating  broth 
cultures  with  B.  pestis,  and  as  soon  as  the  stalactite  formation 
appears  the  tube  is  thoroughly  shaken  until  the  growth  falls 
to  the  bottom.  The  tube  is  then  reinoculated  with  fresh  growth 
and  the  shaking  and  reinoculation  process  is  repeated  five  or  six 
times.  After  about  six  weeks  the  culture  is  killed  by  heating 
for  one  hour  at  65°  C.  The  dose  administered  is  about  3  to  3.5 
c.c.,  an  amount  equivalent  to  about  500  million  bacteria.  Vac- 
cination gives  a  relatively  short  immunity  but  is  sufficiently  marked 
to  warrant  its  use  in  case  of  exposure  to  infection.  In  the  Punjab 
during  the  plague  season  1902-1903,  of  those  inoculated  the  mor- 
tality was  only  23.9  per  cent  as  compared  with  60.1  per  cent 
among  the  uninoculated. 

A  serum  prepared  by  Yersin  by  injecting  horses  first  with  killed 
cultures  and  later  with  living  cultures  of  B.  pestis  is  reported  by 
certain  investigators  to  have  a  curative  action ;  others,  however, 
have  failed  to  secure  any  favorable  result  from  its  use. 


CHAPTER  XXIII 


PATHOGENIC   ANAEROBIC   BACILLI 

B.  Tetani.  —  The  association  of  tetanus  with  wounds  and  soil 
was  early  recognized,  but  for  centuries  all  attempts  to  find  the  excit- 
ing cause  failed.  In  1884  Nicolaier  succeeded  in  infecting  mice 
and  rabbits  by  inoculating  them  with  garden  soil,  but  by  none  of 

the  ordinary  methods  was  he 
able  to  isolate  an  organism 
which  would  give  rise  to  the 
disease.  In  1889  Kitasato  suc- 
ceeded in  isolating  from  lesions 
in  mice  which  had  been  inocu- 
lated with  material  from  a 
human  case  a  bacillus  which 
when  injected  in  pure  cultures 
into  animals  produced  the 
characteristic  symptoms.  He 
further  demonstrated  that  the 
cause  of  the  earlier  failures  to 
obtain  the  organism  was  due  to  the  fact  that  it  could  only  grow 
alone  in  the  absence  of  oxygen. 

Morphology  and  Staining.  —  The  tetanus  bacilli  appear  as 
slender  rods  with  rounded  ends  about  4  /*  in  length  and  0.4  p 
in  diameter.  They  readily  form  spores  which  are  round  and  have 
a  diameter  usually  much  larger  than  the  thickness  of  the  bacilli ; 
the  spore  develops  at  one  end  of  the  organism,  giving  it  an  appear- 
ance somewhat  like  a  drumstick  (Fig.  33).  The  bacilli  are 
slightly  motile  in  the  vegetative  form  and  possess  a  large  number 
of  peritrichal  flagella.  They  stain  with  the  ordinary  anilin  dyes 
and  are  not  decolorized  by  Gram's  method. 

242 


FIG.  33.  —  Tetanus  Bacilli. 


BACILLUS   TETANI  243 

Cultivation.  —  At  20°  to  24°  C.  growth  occurs  slowly  and  spores 
are  produced  in  six  to  ten  days ;  at  37°  C.  development  is  much 
more  rapid  and  spore  formation  begins  within  twenty-four  to  thirty 
hours.  Under  ordinary  conditions  the  tetanus  bacillus  is  a  strict 
anaerobe.  If,  however,  aerobic  bacilli  are  growing  with  it  in  culture 
medium  it  will  develop  even  when  air  is  admitted.  Presumably 
the  air  is  used  up  by  the  associated  aerobes.  Growth  occurs 
abundantly  on  gelatin  or  agar  containing  1  to  2  per  cent  glucose ; 
the  colonies  have  a  fleecy  thread-like  margin  radiating  from  a 
heavier  central  portion.  In  gelatin  stabs  growth  appears  along 
the  needle  track  and  from  it  outgrowths  extend,  giving  the  appear- 
ance of  an  inverted  fir  tree ;  liquefaction  takes  place  slowly,  gen- 
erally with  the  production  of  a  gas  of  characteristic  and  disagree- 
able odor. 

As  the  tetanus  bacillus  is  almost  always  associated  with  other 
organisms  the  most  successful  method  of  obtaining  a  pure  culture 
is  by  taking  advantage  of  the  resistance  of  its  spores  to  heat. 
The  material  containing  the  organism  may  be  inoculated  into  a 
tube  of  glucose  broth  and  incubated  at  37°  C.  for  two  days.  By 
that  time  sporulation  will  have  occurred  and  the  culture  may  then 
be  heated  to  80°  C.  for  three  quarters  of  an  hour  in  order  to  destroy 
the  associated  vegetative  forms.  From  the  heated  culture  agar 
anaerobic  plates  are  made,  and  if  the  tetanus  bacilli  are  the  only 
spore-bearing  anaerobic  organisms  present  a  pure  culture  may  be 
readily  obtained. 

Resistance.  —  In  the  vegetative  form  the  tetanus  bacillus  is 
destroyed  by  the  same  agencies  that  kill  spore-free  bacteria.  On 
the  other  hand,  few  forms  of  life  are  more  resistant  than  tetanus 
spores;  in  a  dried  condition  they  may  retain  their  vitality  for 
years.  In  5  per  cent  carbolic  acid  they  are  killed  in  ten  hours. 
The  addition  of  0.5  per  cent  hydrochloric  acid  hastens  the  germi- 
cidal  effect  and  destroys  them  in  two  hours.  Bichloride  of  mercury 
1  to  1000  kills  them  in  three  hours  and  in  thirty  minutes  if  0.5 
per  cent  hydrochloric  acid  be  added  to  the  solution.  The  spores 
are  completely  destroyed  when  exposed  to  dry  heat  at  160°  C.  for 
one  hour  or  to  steam  at  120°  C.  for  twenty  minutes. 


244  BACTERIOLOGY   FOR  NURSES 

Pathogenesis.  —  Tetanus  under  ordinary  conditions  affects  only 
man  and  horses;  it  can  be  produced,  however,  in  other  animals 
by  the  injection  of  pure  cultures  on  their  toxin. 

The  normal  habitat  of  the  tetanus  bacilli  is  the  intestinal  tract 
of  herbivorous  animals.  In  their  dejecta  the  organisms  find  their 
way  into  the  soil  and  in  the  pulverized  soil  they  are  scattered 
with  the  dust  practically  everywhere.  Tetanus  bacilli  may  be 
found  almost  wherever  man  and  domesticated  animals  have  been. 
In  some  localities  they  are  much  more  numerous  than  in  others, 
and  in  certain  parts  of  Long  Island  and  New  Jersey  an  unusual 
number  of  cases  have  developed  as  a  result  of  small  wounds. 

When  tetanus  bacilli  enter  the  body  by  way  of  the  mouth 
they  are  quite  harmless.  Passing  into  the  intestines,  they  find 
ideal  anaerobic  conditions  and  a  temperature  suitable  to  their 
development,  and  they  are  able  to  multiply  there  without  causing 
any  harm  to  their  host.  It  is  exceedingly  curious  that  the  horse, 
which  may  almost  be  regarded  as  a  tetanus  "  carrier,"  is  the  most 
susceptible  of  all  animals  to  tetanus  toxin.  Rats  and  birds  are 
only  slightly  susceptible  and  fowls  scarcely  at  all.  It  is  estimated 
that  an  amount  of  tetanus  toxin  sufficient  to  kill  a  hen  would  kill 
five  hundred  horses. 

Tetanus  appears  in  man  almost  always  as  a  wound  complica- 
tion, although  the  presence  of  tetanus  spores  in  a  wound  does  not 
necessarily  result  in  infection.  It  has  been  found  by  animal 
experimentation  that  in  a  clean  wound  a  few  spores  free  from  their 
toxin  do  not  give  rise  to  the  disease;  they  are  in  all  probability 
disposed  of  by  the  phagocytes.  On  the  other  hand,  a  lacerated  or 
contused  wound  made  by  a  dirty,  blunt  instrument  is  a  much  more 
favorable  ground  for  their  development,  but  even  in  such  wounds 
if  mixed  infection  is  prevented  by  prompt  disinfection  and  conse- 
quent killing  of  the  associated  bacteria,  tetanus  spores  if  present 
in  all  probability  will  not  develop.  Symbiosis  is  perhaps  the 
main  factor  in  determining  whether  or  not  the  spores  will  be  able 
to  germinate.  The  presence  of  other  organisms,  and  especially  the 
pyogenic  cocci,  seem  to  facilitate  their  development. 

Not  only  traumatic  tetanus  but  all  other  forms  are  now  known 


BACILLUS   TETANI  245 

to  result  from  infection  with  B.  tetani.  In  tetanus  neonatorum 
the  organisms  gain  entrance  through  the  umbilical  wound;  in 
puerperal  cases  through  the  inner  surface  of  the  uterus.  Contam- 
ination of  vaccine  and  sera  used  in  human  therapy  have  unfor- 
tunately occurred.  In  St.  Louis  in  1901  diphtheria  antitoxin 
taken  from  a  horse  during  the  period  of  incubation  of  tetanus 
was  administered  to  seven  children,  all  of  whom  died  of  tetanus. 
Bacteriological  examination  showed  the  serum  to  be  sterile,  but 
it  contained  large  amounts  of  tetanus  toxin.  A  law  has  since  been 
passed  requiring  all  sera  and  vaccines  sold  in  interstate  traffic  to 
be  controlled  by  animal  tests. 

Tetanus  is  a  local  infection  resulting  in  general  toxemia.  Clini- 
cally it  is  characterized  by  a  gradually  increasing  stiffness  and 
spasmodic  contractions  of  the  voluntary  muscles,  commencing 
with  those  of  the  jaw  and  the  back  of  the  neck.  The  spasms 
are  tonic  in  character,  and  as  the  disease  progresses  succeed  each 
other  with  only  a  slight  intermission.  The  bacilli  never  gain  ac- 
cess to  the  blood  and  consequently  are  never  distributed  to  the 
internal  organs  but  remain  localized  at  the  site  of  infection,  where 
they  produce  one  of  the  most  powerful  toxins  known,  which  when 
absorbed  gives  rise  to  the  main  symptoms  and  lesions  of  the  in- 
fection. Therefore  while  the  blood  of  a  tetanus  patient  contains 
no  bacilli  it  is  laden  with  the  toxin  which  is  responsible  for  the 
disease. 

The  most  important  feature  of  tetanus  toxin  is  its  strong  affinity 
for  nerve  tissue.  It  is  rapidly  absorbed  from  the  local  site  of 
infection  into  the  blood  and  lymph  streams,  is  distributed  to  the 
muscles,  and  it  is  thought  by  many  investigators  reaches  the 
spinal  cord  and  brain  indirectly  through  absorption  by  the  end 
plates  of  the  motor  nerves. 

Regardless  of  the  amount  of  infection  there  is  always  an  incuba- 
tion period  during  which  the  bacilli  multiply  and  produce  the  toxin. 
Generally  speaking,  the  severer  the  infection  the  shorter  will  be 
the  period  of  incubation  and  the  greater  the  probability  that  the 
case  will  have  a  fatal  ending.  The  toxin  is  produced  and  may  be 
absorbed  during  or  soon  after  the  first  twenty-four  hours  follow- 


246  BACTERIOLOGY   FOR  NURSES 

ing  infection ;  hence  the  necessity  for  the  immediate  administra- 
tion of  antitoxin.  For,  once  the  toxin  has  entered  into  a  firm  union 
with  the  nerve  cells  it  cannot  be  displaced.  Antitoxin  in  sufficient 
amounts  will  neutralize  the  toxin  as  quickly  as  it  is  produced  and 
thus  protect  the  nerve  tissue  until  the  leukocytes  and  other  body 
cells  have  destroyed  the  bacilli  and  spores.  Unfortunately  treat- 
ment is  often  deferred  until  symptoms  have  appeared.  In  such 
cases  all  the  antitoxin  can  do  is  to  combine  with  the  free  toxin 
and  prevent  further  damage.  As  a  therapeutic  measure  injec- 
tions are  usually  made  intravenously  or  by  means  of  a  lumbar 
puncture  in  order  that  the  effect  may  be  as  speedy  as  possible. 
In  acute  cases  50,000  to  100,000  units  are  administered  during 
the  first  few  days.  A  prophylactic  dose  is  usually  1000  units 
injected  subcutaneously  or  intramuscularly. 

The  method  of  preparing  tetanus  antitoxin  for  the  purpose 
of  passive  immunization  is  described  in  Chapter  II. 

Bacillus  Welchii  (B.  Aerogenes  Capsulatus).  —  The  name  applies 
rather  to  a  group  than  to  an  individual  organism.  Welch  and 
Mittal  in  1892  were  the  first  to  isolate  and  describe  minutely  a 
member  of  the  group,  and  they  gave  to  the  organism  the  name 
B.  aerogenes  capsulatus.  It  is  frequently  spoken  of,  however,  as 
the  Welch  bacillus.  Strains  of  bacilli  closely  related  to  and  prob- 
ably identical  with  the  Welch  bacillus  have  been  described,  among 
which  are  B.  phlegmonis  emphysematosi,  B.  perfringens,  B. 
enteritidis  sporogenes,  and  a  number  of  others.  During  the  recent 
war  the  bacilli  have  been  recognized  as  the  most  important  agents 
in  the  production  of  gas  gangrene  in  infected  wounds. 

Morphology  and  Staining.  —  B.  Welchii  is  a  comparatively 
large  bacillus  measuring  from  4  to  6  ft  in  length  and  relatively 
thick.  In  preparations  from  tissues  or  body  fluids  a  distinct 
capsule  is  seen  surrounding  it ;  hence  its  original  name.  Spores 
are  formed  by  some  strains  and  are  most  likely  to  appear  when 
blood  serum  is  used  as  a  medium. 

Cultivation.  —  On  agar  the  colonies  are  round  with  smooth 
margins  and  no  outgrowths.  The  bacillus  ferments  glucose, 
saccharose,  lactose,  and  maltose  with  the  production  of  acid  and 


BACILLUS   OF   MALIGNANT   EDEMA  247 

gas.  It  does  not  liquefy  gelatin.  In  milk  its  growth  is  especially 
characteristic  :  acid  is  rapidly  formed,  coagulation  occurs,  and  soon 
the  clot  becomes  torn  apart  by  gas  bubbles,  so-called  "  stormy 
fermentation."  Ultimately  it  forms  an  irregular  firm  mass  in 
the  comparatively  clear  whey.  The  organism  is  a  strict  anaerobe. 
Growth  will  occur  at  room  temperature,  but  is  most  abundant  at 
37°  C. 

Pathogenesis.  —  In  the  lower  animals  infection  seldom  occurs. 
In  man  the  organism  has  been  observed  in  diseases  of  the  gastro- 
intestinal and  genito-urinary  tracts.  When  such  cases  end  fatally 
the  bacilli  frequently  pass  into  the  blood  stream  at  the  time  of 
death  and  produce  gas  cavities  in  the  various  internal  organs ;  the 
so-called  "  foamy  organs  "  observed  at  autopsy.  The  bacillus 
is  of  special  interest  in  that  it  has  been  by  far  the  most  important 
cause  of  gas  gangrene  in  war  wounds.  Laceration  of  the  muscle 
tissue  with  an  object  contaminated  with  soil  containing  the  bacillus 
is  usually  the  starting  point  of  infection.  The  remarkable  feature 
of  the  disease  is  the  rapidity  with  which  it  spreads.  Cases  have 
been  recorded  in  which  emphysematous  swelling  and  gangrene  of 
a  limb  has  ended  fatally  within  twenty-four  hours. 


BACILLUS   OF  MALIGNANT  EDEMA 

The  organism  was  first  discovered  by  Pasteur  in  1877  in  putrid 
flesh.  He  found  that  by  introducing  such  flesh  into  rabbits  an 
edematous  condition  of  the  tissues  and  degenerative  changes  in 
the  various  organs  were  produced.  Koch  and  Gaffky  in  1881 
isolated  the  organism,  carefully  studied  it,  and  gave  to  it  the  name 
B.  edematis  maligni.  The  organism  is  found  in  the  intestinal 
tract  of  the  higher  animals,  in  the  upper  layer  of  the  soil  in  putre- 
fying substances,  and  in  polluted  water.  During  the  recent  war 
it  was  found  in  putrid  wounds  and  cases  of  gas  gangrene  next  in 
order  of  frequency  to  B.  Welchii. 

Morphology  and  Staining.  —  The  bacilli  vary  in  length  from  2 
to  10  p  and  0.8  to  1  /A  in  width.  They  are  usually  seen  in  pairs 
joined  end  to  end ;  occasionally  they  occur  in  long  chains.  The 


248  BACTERIOLOGY  FOR  NURSES 

organism  is  motile  and  forms  an  oval  spore  situated  in  the  center 
of  the  bacillus.  It  is  stained  readily  by  the  ordinary  dyes.  Young 
cultures  are  usually  Gram  positive ;  older  ones  appear  to  be  less 
able  to  retain  the  stain. 

Cultivation . — The  organism  is  a  strict  anaerobe .  Growth  occurs 
at  20°  C.,  but  is  more  rapid  and  abundant  at  37°  C.  On  glucose 
agar  colonies  appear  of  a  dull  white  color  with  an  irregular  margin. 
It  is  able  to  ferment  glucose,  lactose,  and  maltose  and  to  liquefy 
gelatin. 

Pathogenesis.  —  A  number  of  animals  are  susceptible  to  inocu- 
lation with  the  bacilli.  Subcutaneous  injections  produce  a  spread- 
ing gelatinous  edema  and  exudation  of  a  blood-stained  fluid,  while 
the  underlying  muscles  become  soft  and  partly  necrosed.  At- 
tempts to  produce  the  disease  by  feeding  animals  with  the  bacilli 
or  by  injecting  them  intravenously  have  so  far  failed.  In  man 
practically  all  infections  appear  to  have  started  from  fractured 
bones  or  deep  wounds. 

BACILLUS   CHAUVEI 

Symptomatic  anthrax,  popularly  known  as  "  black  leg "  or 
"  quarter  evil,"  is  an  infectious  disease  occurring  chiefly  among 
cattle  and  sheep ;  so  far  as  is  known  infection  has  never  occurred 
in  man.  The  disease  was  formerly  confused  with  true  anthrax. 
The  microorganisms  which  give  rise  to  the  two  diseases,  however, 
are  totally  different.  Morphologically  and  culturally  B.  chauvei 
closely  resembles  the  bacilli  of  malignant  edema.  The  disease  is 
usually  rapidly  fatal.  Edematous  swelling  on  the  thigh  or  shoulder 
appears,  in  a  few  hours  a  considerable  quantity  of  gas  collects  in 
the  tissues,  and  the  affected  muscles  become  almost  black.  Death 
usually  occurs  in  from  one  to  two  days.  The  disease  is  usually 
due  to  wound  infection  with  soil  containing  the  bacilli. 

Differentiation  of  Wound  Anaerobes.  —  Practically  all  of  the 
anaerobes  concerned  in  wound  infections  come  from  the  soil  and 
originally  from  animal  feces.  The  group  appears  to  be  closely 
related.  The  following  characteristic  points  of  each,  however,  are 
relatively  constant  and  afford  a  means  of  differentiation. 


BACILLUS  BOTULINUS  249 

B.  Tetani  presents  a  drumstick  appearance  due  to  large  terminal 
spores ;  in  glucose  stab  cultures  growth  resembles  an  inverted  tree. 

B.  Welchii.  —  One  of  the  surest  tests  for  differentiating  B. 
Welchii  from  the  other  anaerobes  was  devised  by  Welch  and  Mit- 
tal.  Bacilli  are  injected  into  the  ear  vein  of  a  rabbit,  which  is 
killed  after  a  few  minutes  by  a  blow  on  the  head.  The  body  is 
incubated  and  within  twenty-four  hours  it  becomes  tensely  dis- 
tended with  gas,  and  at  autopsy  gas  bubbles  are  found  in  all  the 
organs. 

B.  Edematis  may  be  distinguished  from  B.  Welchii  by  the  fact 
that  it  is  motile  and  the  lesions  it  produces  are  edematous  rather 
than  emphysematous. 

B.  Chauvei.  —  Injection  of  pure  cultures  into  rabbits  is  the 
best  means  of  differentiating  B.  chauvei  from  B.  edematis  and 
B.  welchii.  Rabbits  are  immune  to  the  former  and  susceptible 
to  the  two  latter  organisms. 

BACILLUS  BOTULINUS 

As  already  stated  the  term  meat  poisoning  is  applied  to  dif- 
ferent conditions  produced  by  different  agents.  The  relation  of 
certain  members  of  the  colon-typhoid  group  to  so-called  ptomain 
poisoning  has  already  been  noted.  Another  and  totally  different 
type  of  food  poisoning  is  due  to  the  toxin  generated  by  B.  botulinus 
during  its  growth  on  nitrogenous  substances  outside  of  the  body. 
The  bacillus  is  a  parasite  and  does  not  multiply  within  the  body. 
Fortunately  it  requires  time  for  it  to  develop  and  produce  its  toxin, 
and  for  this  reason  fresh  foodstuffs  are  not  apt  to  be  dangerous  so 
far  as  botulism  is  concerned.  Sausage,  canned  meat,  and  fish  have 
been  found  mainly  responsible  for  the  conveyance  of  the  poison  to 
human  beings.  It  is  of  singular  importance  that  meat  may  con- 
tain numbers  of  the  bacilli  and  relatively  large  amounts  of  the 
poison  without  any  visible  sign  of  decomposition.  In  1896  van 
Ermengen  isolated  B.  botulismus  from  a  sample  of  ham  which 
had  been  eaten  raw  and  which  had  caused  a  number  of  cases  of 
poisoning,  some  of  which  had  ended  fatally. 


250  BACTERIOLOGY  FOR  NURSES 

Morphology  and  Staining.  —  The  organism  is  a  large  bacillus 
measuring  from  4  to  9  /A  in  length  and  .9  to  1.2  in  width.  It 
forms  large  terminal  spores,  is  motile,  and  Gram  positive. 

Cultivation.  —  Colonies  are  yellow  and  coarsely  granular, 
gelatin  is  liquefied  and  glucose  is  fermented  with  the  production 
of  acid  and  gas.  The  organism  is  a  strict  anaerobe  growing  best 
at  22  to  24°  C. ;  it  does  not  multiply  at  a  temperature  above  35°  C. 
Hence  growth  does  not  take  place  in  the  body. 

Pathogenesis.  —  The  poison  produced  by  B.  botulinus  is  a 
true  toxin  in  the  same  sense  as  that  of  diphtheria  or  tetanus.  It 
is,  however,  entirely  produced  outside  of  the  body.  Thus  the 
bacillus  occupies  an  unique  position  in  that  it  forms  its  poison  only 
on  dead  nitrogenous  substances. 

Unlike  the  toxin  of  diphtheria  or  tetanus  it  is  poisonous  when 
taken  by  mouth,  absorption  evidently  taking  place  from  the  in- 
testinal canal.  Symptoms  of  botulism  appear  after  ingestion  of 
the  poison  in  from  twenty-four  to  forty-eight  hours  and  are  chiefly 
the  effect  of  the  toxin  on  the  cranial  nerves.  Dilated  pupils,  dys- 
phagia,  and  sometimes  aphagia  and  aphonia,  profuse  secretion  from 
the  mouth  and  nose,  constipation  and  retention  of  urine,  and  more 
or  less  derangement  of  the  cardiac  and  respiratory  centers  are 
apt  to  occur. 

The  toxin  is  pathogenic  for  several  of  the  lower  animals.  It 
is  readily  destroyed  by  heat ;  it  also  deteriorates  rather  quickly  in 
sunlight. 

Botulism  differs  mainly  from  the  "  meat-poisoning  "  due  to 
members  of  the  colon-typhoid  group  in  that  the  poison  produced 
is  a  true  toxin  and  causes  the  production  of  an  antitoxin.  The 
causal  agent  cannot  multiply  in  the  body  or  in  the  presence  of 
oxygen.  Few  or  no  lesions  occur  in  the  intestinal  canal. 

B.   FUSIFORMIS 

The  organism  has  been  observed  to  be  constantly  present  in 
large  numbers  in  pseudomembranous  conditions  of  the  mouth  and 
throat  known  as  "  ulceromembranous  angina "  or  "  Vincent's 


TYPHUS    FEVER 


251 


angina,"  and  because  of  this  fact  it  is  strongly  suspected  as  the 
etiological  cause  of  the  disease.  The  bacillus  is  a  long,  slender 
organism,  slightly  curved,  pointed  at  both  ends,  and  somewhat 
swollen  in  the  center,  presenting  thus  a  spindle-shaped  or  fusiform 
appearance  (Fig.  34).  It  is  non-motile  and  Gram  negative. 
Grown  under  anaerobic  conditions  it  forms  a  whitish  sediment  in 
broth ;  on  agar  the  colonies 
have  characteristic  filament- 
ous outgrowths. 

In  film  preparations  made 
directly  from  a  lesion  long 
spirilla  seem  to  be  almost 
as  numerous  as  the  fusiform 
bacilli.  When,  however,  cul- 
ture medium  is  inoculated  only 
fusiform  bacilli  can  be  found. 
The  fact  has  given  rise  to 
much  speculation  as  to  the 
relation  of  the  two  forms. 
Many  investigators  have  con- 
sidered the  two  organisms  as  distinct,  but  as  having  a  symbiotic 
affinity ;  others  have  advanced  the  theory  that  the  two  forms  are 
only  different  phases  in  the  life  history  of  one  organism. 

Fusiform  bacilli  associated  with  spirilla  have  also  been  found 
in  gangrene,  noma,  gingivitis,  and  dental  caries. 


FIG.  34.  —  Fusiform  Bacilli  and  Spiro- 
chetes. 


TYPHUS   FEVER 

The  disease,  said  by  various  writers  to  be  one  of  the  most  highly 
contagious  of  all  febrile  diseases,  occurs  principally  under  conditions 
of  overcrowding  and  filth.  Formerly  it  appeared  in  epidemic 
form.  Improved  sanitation,  however,  has  greatly  diminished  its 
occurrence ;  and  in  some  localities  it  has  practically  disappeared. 
Infection  is  characterized  by  an  incubation  period  of  from  five  to 
ten  days,  followed  by  a  high  temperature  and  petechial  rash. 
The  body  louse  seems  to  be  mainly  responsible  for  its  transmis- 


252  BACTERIOLOGY  FOR  NURSES 

sion  and  in  all  probability  the  head  louse  too,  a  fact  which  justifies 
its  classification  as  a  filth  disease.  Typhus  fever  still  occurs  in 
certain  parts  of  Europe  and  in  North  and  South  America.  In 
Mexico  it  appears  epidemically  under  the  name  of  Tarbardillo. 
An  infection  known  as  Brill's  disease  and  thought  by  Brill  to  be  a 
new  malady  has  recently  been  shown  to  be  a  mild  form  of  typhus 
fever. 

For  a  long  time  the  disease  has  been  attributed  to  a  filtrable 
virus,  and  some  observers  still  hold  the  opinion.  Many  extensive 
investigations  have  been  made  as  to  its  etiology,  and  various 
organisms  have  been  described  as  the  probable  causal  agent, 
but  until  quite  recently  all  attempts  to  obtain  cultures  failed. 
In  1914  Plotz  obtained  from  the  blood  of  cases  of  Brill's  disease 
an  organism  which  he  succeeded  in  cultivating  anaerobically. 
In  a  great  many  subsequent  cases  Plotz  and  his  co-workers  have 
found  the  same  organism  and  have  demonstrated  the  presence  of 
agglutinins  and  complement-fixing  antibodies  in  the  blood  of 
convalescents.  Much  of  the  evidence  so  far  obtained  is  in  favor 
of  the  bacillus  as  the  causal  agent  of  typhus  fever. 


CHAPTER  XXIV 


IN  the  Delta  of  the  Ganges  Asiatic  cholera  has  been  known  for 
centuries.  Not  until  the  nineteenth  century,  however,  did  it  appear 
in  Europe  and  America.  Traveling  along  the  trade  route  it  reached 
Europe  in  1830,  and  in  1832  it  arrived  in  America  by  way  of  New 
York  and  Quebec  and  spread  west  as  far  as  the  Mississippi.  By 
1849  it  had  traveled  with  the  searchers  for  gold  as  far  as  California. 

Prior  to  1883  nothing  was  known  regarding  the  causal  agent ; 
in  that  year  Koch  discovered  in  the  feces  of  cholera  patients  a 
curved  organism  now  generally 
known  as  the  "comma  bacil- 
lus "  or  "  cholera  spirillum." 
Soon  other  observers  obtained 
comma-shaped  organisms  from 
many  other  sources,  and  a 
great  deal  of  controversy  arose 
as  to  their  classification.  With 
the  advent  of  serological  tests, 
however,  all  of  these  organisms 
except  the  vibrio  of  Koch  were 
shown  to  be  unaffected  by  the 

»         .        ,  .       ,  FIG.  35.  —  Cholera  Spirilla. 

serum  ot  animals  immunized 

to  cholera  and  hence  in  no  way  connected  with  the  disease. 

Morphology  and  Staining.  —  The  cholera  spirillum  appears  in 
stained  preparations  as  a  curved  rod  about  1.5  fi  in  length  and 
0.4  /u  in  width.  A  single  organism  may  appear  slightly  curved 
like  a  comma.  Two  organisms  remaining  attached  and  curved  in 
an  opposite  direction  may  produce  a  resemblance  to  the  letter  S, 
while  adherent  in  greater  numbers  they  may  appear  as  a  long, 
spiral  filament  (Fig.  35).  The  organism  is  actively  motile  although 

253 


254 


BACTERIOLOGY  FOR  NURSES 


FIG.  36.  — Cholera  Spirilla,  enlarged  to 
show  Flagella. 


it  possesses  only  a  single  long  fine  flagellum  attached  to  one 
end  (Fig.  36).  Small  highly  refractile  bodies  are  sometimes  noted, 
but  true  spores  are  not  formed.  Staining  is  best  accomplished 
with  Loeffler's  methylene  blue  or  a  weak  solution  of  carbol 

fuchsin.     The  organisms  are 
Gram  negative. 

Cultivation.  —  The  cholera 
spirillum  is  an  aerobe  and 
grows  on  all  ordinary  media 
provided  the  reaction  is  dis- 
tinctly alkaline  to  litmus.  De- 
velopment is  most  luxuriant  at 
37°  C.  although  multiplication 
will  occur  at  a  temperature  as 
low  as  16°  C.  In  gelatin  stab 
cultures  growth  appears  along 
the  needle  tract,  and  in  about 
twenty-four  hours  liquefaction 
commences  on  the  surface  as  a  small  bell-shaped  area  which 
deepens  until  by  the  end  of  the  week  the  entire  medium  may  be 
fluid.  On  gelatin  plates  the  colonies  appear  as  minute  whitish 
points  with  a  granular  surface  somewhat  resembling  a  layer  of 
powdered  glass.  After  twenty-four  to  forty-eight  hours  liquefac- 
tion of  the  gelatin  commences  around  each  colony  and  as  it  pro- 
gresses the  colony  sinks  to  the  bottom  of  the  cup-like  depression 
thus  formed.  On  agar  the  colonies  have  a  characteristic  opalescent 
appearance ;  on  potato  growth  takes  place  only  at  37°  C.  and  then 
it  appears  as  a  dirty  moist  layer.  Acid  is  rapidly  produced  from 
glucose,  saccharose,  and  maltose ;  coagulated  blood  serum  is  lique- 
fied. In  milk  growth  takes  place  without  producing  any  visible 
change. 

Indol  is  produced  in  peptone  water  medium  by  all  true  cholera 
spirilla ;  certain  other  organisms  have  the  same  ability,  but  they 
are  comparatively  few.  The  cholera  spirillum  produces  nitrites 
at  the  same  time  as  indol,  so  that  in  applying  the  test  it  is  only 
necessary  to  add  a  few  drops  of  sulphuric  acid  to  a  peptone  water 


CHOLERA  SPIRILLUM  255 

culture  which  has  been  incubated  twenty-four  to  forty-eight 
hours.  When  the  growth  is  that  of  a  cholera  spirillum  a  reddish 
pink  color  appears,  the  so-called  "  cholera  red  reaction." 

Resistance.  —  The  cholera  spirillum  has  much  the  same  degree 
of  resistance  that  other  spore-free  organisms  possess.  It  is  killed 
by  exposure  to  moist  heat  at  60°  C.  in  ten  minutes  and  at  100°  C. 
in  one  minute.  Chemical  disinfectants  are  speedily  effective. 
The  organisms  are  especially  sensitive  to  drying,  hence  it  is  unlikely 
they  are  ever  carried  in  a  living  condition  in  the  dust  particles 
in  the  air.  In  the  dejecta  of  cholera  patients  they  remain  alive 
usually  from  one  to  three  days,  although  occasionally  they  have 
been  found  after  a  much  longer  period.  In  running  water  they 
persist  for  six  or  seven  days,  and  in  stagnant  water  for  about  eigh- 
teen days.  In  milk  the  acidity  produced  by  other  bacteria  soon 
destroys  them. 

Modes  of  Transmission.  —  Cholera  spirilla  leave  the  body  of 
infected  patients  in  the  feces  and  so  far  as  is  known  enter  only  by 
way  of  the  mouth.  Consequently,  food  and  water,  directly  or  in- 
directly contaminated  with  the  dejecta  of  infected  individuals, 
are  the  main  cause  of  the  spread  of  the  disease.  Water  is  probably 
solely  responsible  for  the  great  epidemic  outbursts. 

The  earliest  authentic  account  of  an  epidemic  of  cholera  trace- 
able to  polluted  water  is  the  Broad  Street  pump  case  in  London 
in  1854.  Within  five  weeks  616  deaths  occurred  in  the  vicinity 
amongst  people  who  drank  of  the  water.  The  outbreak  happened 
before  the  days  of  bacteriology,  consequently  absolute  proof  of 
the  presence  of  the  cholera  spirillum  was  not  available.  Inspection 
of  the  premises,  however,  revealed  a  cesspool  with  defective  brick- 
work from  which  fluid  material  was  constantly  percolating  into 
the  well. 

In  Hamburg  in  1892  a  similar  explosive  outbreak  occurred. 
Cholera  was  taken  to  the  city  by  immigrants  and  the  water  of  the 
Elbe  was  infected  with  their  discharges.  The  sewers  of  Hamburg 
emptied  into  the  river  near  the  water  intake  from  which  the  city 
received  its  supply  for  drinking  purposes.  The  adjoining  city  of 
Altona  received  its  water  from  the  same  source,  but  purified  it  by 


256  BACTERIOLOGY  FOR  NURSES 

sand  filtration  before  use.  Throughout  the  period  of  the  epidemic 
houses  situated  on  one  side  of  a  street  and  supplied  with  Hamburg 
water  developed  many  cases  of  cholera,  while  those  on  the  other 
side  of  the  street  receiving  their  supply  from  Altona  remained 
uninfected. 

Epidemics  occasionally  occur  which  are  more  difficult  to  trace 
to  their  source.  Cholera  spirilla  have  been  found  in  the  feces  of 
healthy  individuals  and  of  people  suffering  from  slight  intestinal 
disturbances.  "  Cholera  carriers  "  therefore  are  a  possible  foci  of 
infection.  Investigations  have  shown  that  the  spirilla  tend  to 
disappear  from  the  feces  in  from  four  to  fourteen  days.  They 
have  been  known,  however,  to  persist  for  sixty-nine  days  and 
longer.  Contact  infection  plays  an  important  role  where  persons 
live  together  under  uncleanly  conditions.  The  careless  handling 
of  dejecta  and  soiled  linen  is  especially  liable  to  result  in  infection. 
Haffkine,  in  India,  found  that  sterilized  milk  if  left  in  open  jars 
to  which  flies  had  access  might  become  contaminated  with  cholera 
organisms  in  a  cholera-infected  locality. 

Pathogenesis.  —  Asiatic  cholera  appears  to  be  a  disease  peculiar 
to  man ;  none  of  the  lower  animals  have  been  known  to  contract  it 
naturally.  Intraperitoneal  injections  of  the  organisms  into  a 
guinea  pig  may  be  speedily  fatal,  but  intestinal  lesions  are  rarely 
seen.  Koch  succeeded  in  producing  the  disease  in  much  the  same 
form  as  it  appears  in  man  by  first  neutralizing  the  gastric  juice 
with  a  solution  of  carbonate  of  soda  and  inhibiting  peristalsis  by 
an  injection  of  tincture  of  opium  and  then  introducing  a  culture 
of  the  cholera  spirilla  into  the  intestinal  tract  by  means  of  a  cath- 
eter. Metchnikoff  obtained  similar  results  with  new-born  rabbits 
by  rubbing  a  small  amount  of  culture  on  the  teats  of  the  mother 
rabbit. 

In  man  the  lesions  are  primarily  intestinal.  The  short  incuba- 
tion period,  which  is  usually  one  to  two  days  and  rarely  over  five, 
is  indicative  of  the  rapid  multiplication  of  the  organisms  once 
they  have  gained  entrance  to  the  alimentary  tract,  and  to  the 
production  of  a  speedily  effective  toxin.  The  lower  part  of  the 
small  intestine  is  the  part  most  affected.  Penetrating  the  surface 


CHOLERA  SPIRILLUM  257 

of  the  mucosa  the  organisms  loosen  the  epithelial  cells,  which 
are  shed  in  flakes  and  give  to  the  stools  their  characteristic  rice- 
water  appearance.  In  the  more  chronic  forms  of  the  disease 
extensive  necrosis  of  the  intestinal  wall  and  the  formation  of  a 
false  membrane  may  occur  together  with  a  considerable  amount 
of  hemorrhage.  It  is  generally  thought  that  the  organisms  never 
invade  the  blood  stream  and  internal  organs. 

The  causal  relation  of  the  "  comma  bacillus  "  of  Koch  to  Asiatic 
cholera  has  been  fully  established  by  a  number  of  laboratory 
experiments  and  accidents.  In  1884  a  student  in  Koch's  labor- 
atory in  Berlin  suddenly  developed  a  severe  attack  of  cholera, 
and  infection  could  have  come  in  no  other  way  than  through  the 
cholera  cultures  with  which  the  man  had  been  working.  In  another 
German  laboratory  Pettenkoffer  and  Emmerich  experimented 
upon  themselves  by  swallowing  a  small  quantity  of  a  fresh  cholera 
culture.  Pettenkoffer  developed  a  mild  attack  of  the  disease,  but 
Emmerich  became  seriously  ill.  In  both  cases  numerous  cholera 
spirilla  were  found  in  the  stools.  Dr.  Oergal,  an  assistant  in  the 
Hamburg  Hygienic  Institute,  became  accidentally  infected  while 
experimenting  with  the  peritoneal  fluid  of  an  injected  guinea  pig. 
After  a  few  days  he  died  of  typical  cholera,  although  there  were 
no  other  cases  in  the  city  at  the  time. 

On  the  other  hand,  many  similar  experimental  cases  have  given 
negative  results.  This,  however,  may  be  explained  as  due  to  differ- 
ent degrees  of  susceptibility.  The  positive  cases  have  been  suffi- 
ciently well  marked  to  warrant  the  acceptance  of  the  organism 
as  the  causal  agent  of  the  disease. 

Immunity.  —  One  attack  of  cholera  produces  a  moderate  degree 
of  immunity  of  rather  short  duration.  Prophylactic  vaccination 
affords  a  certain  degree  of  protection  in  case  of  exposure  to  the 
disease. 

Bacteriological  Diagnosis.  —  A  stained  film  preparation  or  a 
hanging  drop  is  made  from  the  feces  and  examined  microscopically. 
In  some  cases  the  spirilla  are  so  numerous  and  present  a  picture 
so  unique  that  a  microscopic  examination  is  sufficient  for  diagnosis 
during  an  epidemic.  For  the  detection  of  carriers  or  in  case  of 


258       .  BACTERIOLOGY  FOR  NURSES 

the  first  appearance  of  a  cholera-like  disease  other  tests  are  applied. 
Usually  peptone  water  is  inoculated  with  a  small  amount  of  feces, 
and  at  the  end  of  six  to  twelve  hours  a  hanging  drop  is  made  from 
the  surface  growth.  If  the  organisms  are  sufficiently  numerous 
agglutination  tests  are  made  with  the  serum  of  an  immunized 
animal.  Control  tests  are  made  at  the  same  time  with  a  known 
cholera  strain.  A  speedy  method  for  the  detection  of  suspected 
cases  when  a  number  of  examinations  must  be  made  is  the  inocu- 
lation with  feces  of  saccharose  peptone  water  to  which  an  indi- 
cator has  been  added.  As  the  cholera  vibrio  has  the  ability 
to  ferment  saccharose,  decolorization  occurs  in  from  five  to  eight 
hours.  The  tubes  not  decolorized  may  be  discarded,  and  no  further 
examination  is  necessary  since  the  cholera  spirillum  is  not  present. 
Because  of  the  presence  of  sugar  the  decolorized  cultures  are 
unsuitable  for  agglutination  tests.  The  difficulty,  however,  is 
avoided  and  time  saved  if  duplicate  inoculations  are  made,  one  in 
saccharose  medium  and  the  other  in  plain  peptone  water.  In  this 
way  the  peptone  culture  corresponding  to  the  decolorized  saccharose 
culture  is  used  for  the  agglutination  test  as  confirmatory  evidence. 
By  this  method  a  great  many  unnecessary  microscopic  tests  can 
be  eliminated  and  a  diagnosis  made  of  a  large  number  of  cases  in 
a  few  hours. 

Allied  Spirilla.  —  El  Tor  Vibrios.  —  Six  different  strains  of 
spirilla  were  isolated  by  Gotschlich  at  El  Tor  from  the  bodies 
of  pilgrims  on  the  way  to  Mecca,  who  had  died  with  dysenteric 
symptoms  although  there  were  no  cases  of  cholera  in  the  vicinity. 
These  El  Tor  strains  appear  identical  with  the  cholera  spirilla 
morphologically  and  culturally  and  in  their  serological  relation, 
but  in  addition  they  produce  a  strong  hemolysin.  There  is  still 
a  difference  of  opinion  as  to  whether  they  should  be  classed  with 
the  true  cholera  or  regarded  as  a  distinct  species. 

Spirillum  Metchnikovii.  —  The  organism  was  first  obtained 
by  Gamaleia  from  a  cholera-like  disease  of  fowls  epidemic  in 
Odessa.  Morphologically  and  culturally  it  is  identical  with  the 
cholera  spirillum  save  that  colonies  on  agar  have  a  brownish  tinge. 
It  can  readily  be  distinguished  from  the  latter,  however,  by  serum 


SPIROCHETES  259 

reaction  and  by  pigeon  inoculations.  A  minute  amount  of  a  culture 
of  S.  Metchnikovii  injected  subcutaneously  into  a  pigeon  gives 
rise  to  a  rapidly  fatal  septicemia ;  the  same  quantity  of  a  cholera 
culture  produced  practically  no  effect. 

S.  Massaval  and  S.  Finkler-Prior,  both  isolated  from  feces,  and 
S.  Deneke  isolated  from  cheese,  all  closely  resemble  the  cholera 
bacillus,  but  they  do  not  give  a  specific  reaction  with  cholera- 
immune  serum. 

SPIROCHETES 

Because  the  structure  of  certain  spiral  organisms  appears  to 
be  more  complicated  than  that  of  many  bacterial  forms,  and  be- 
cause several  observers  have  found  structural  similarities  between 
them  and  the  protozoa,  they  have  come  to  be  regarded  by  many 
bacteriologists  as  members  of  the  latter  group,  or  as  a  separate 
genus  intermediate  between  bacteria  and  protozoa.  Their  classi- 
fication, however,  is  still  undecided.  The  discovery  that  a  spiro- 
.  chete  was  the  cause  of  syphilis  brought  the  organisms  into  great 
prominence,  and  since  then  many  varieties  have  been  isolated  and 
studied.  The  diseases  produced  by  them  fall  into  two  main  groups  : 
one  usually  transmitted  by  contact  and  in  which  infection  is  pri- 
marily of  the  tissues,  as  in  syphilis  and  yaws;  and  the  second, 
a  blood  infection  accompanied  by  fever  and  transmitted  by  an 
animal  parasite. 

Treponema  Pallidum  (Spirocheta  pallida) . 

Schaudinn  and  Hoffman,  working  together  in  1905,  found  in  the 
fresh  exudates  of  syphilitic  lesions  a  spirochete  which  they  thought 
might  be  the  cause  of  the  disease  and  to  which  they  gave  the  name 
Spirocheta  pallida.  Later  they  decided  that  the  organism  was 
sufficiently  distinctive  to  be  placed  in  a  separate  genus  and  they 
changed  the  name  to  Treponema  pallidum. 

Morphology  and  Staining.  —  The  organism  appears  as  a  long, 
slender  spiral  averaging  about  10  p  in  length  and  0.3  //.  in  diam- 
eter and  with  three  to  twenty  small,  sharp,  regular  curves.  The 
ends  are  pointed  and  at  each  is  a  fine  flagellum  (Fig.  37) .  Movement 
may  be  of  gliding  to-and-fro  nature,  rotation  on  the  long  axis,  or 


260 


BACTERIOLOGY  FOR  NURSES 


FIG.  37.  —  Treponema  Pallidum. 


bending  of  the  entire  body.  It  is  thought  by  some  observers  that 
division  takes  place  in  a  longitudinal  rather  than  a  transverse  direc- 
tion, as  among  many  of  the  protozoa.  The  organisms  are  seen  with 
difficulty  in  unstained  preparations  by  ordinary  microscopic 
methods.  Their  presence  in  smears  is  best  demonstrated  by  the 

Indian  ink  method  and  in 
tissue  sections  by  the  silver 
impregnation  method. 

Cultivation.  —  Schereschew- 
sky  was  the  first  to  cultivate  T. 
pallidum  artificially,  although 
never  in  pure  cultures.  In 
1911  Noguchi  obtained  the 
organisms  from  syphilitic 
lesions  and  succeeded  in  culti- 
vating them  in  the  following 
manner.  The  medium  em- 
ployed consisted  of  one  part 
of  ascitic  or  hydrocele  fluid  and  two  parts  of  2  per  cent  agar  to 
which  was  added  a  small  piece  of  sterile  rabbit  kidney  or  other 
organ.  The  medium  was  covered  by  a  deep  layer  of  paraffin  oil 
in  order  that  strict  anaerobic  conditions  might  be  maintained,  and 
a  stab  inoculation  with  the  material  containing  the  spirochetes 
was  made  through  the  oil  into  the  medium  beneath.  After  ten 
days'  incubation  the  spirochetes  were  found  to  have  grown  out 
into  the  surrounding  medium,  while  most  of  the  associated  organ- 
isms remained  along  the  needle  track.  Subcultures  made  from 
the  outgrowths  finally  resulted  in  a  pure  culture  of  T.  pallidum. 

Pathogenesis.  —  Infection,  so  far  as  known,  never  occurs  among 
the  lower  animals  except  as  a  result  of  inoculation  with  pure 
cultures  or  material  containing  the  human  virus.  By  this  means 
investigators  have  recently  succeeded  in  producing  the  disease 
both  in  rabbits  and  monkeys. 

In  man  the  disease  is  acquired  by  direct  contact  with  infected 
persons  or  things.  It  runs  a  chronic  course,  usually  divided  into 
three  stages,  primary,  secondary,  and  tertiary.  The  initial  or 


TREPONEMA   PALLIDUM  261 

primary  lesion  appears  two  or  three  weeks  after  infection,  first 
as  a  papule  which  develops  into  an  ulcer  with  a  hardened  base, 
the  so-called  chancre,  and  at  the  same  time  there  is  a  marked 
swelling  of  the  nearest  lymph  nodes.  The  symptoms  subside  and 
six  or  seven  weeks  later  secondary  lesions  appear  as  an  eruption 
on  the  skin  and  mucous  membranes,  accompanied  by  general 
constitutional  disturbances.  In  the  tertiary  stage  masses  of  new 
tissue,  spoken  of  as  gummata,  are  formed  through  the  viscera  and 
in  the  periosteum.  The  organisms  may  usually  be  found  in  great 
numbers  in  the  primary  sore  and  in  the  papules  and  mucous  patches 
which  appear  during  the  secondary  stage.  The  latter  fact  explains 
the  infectiousness  of  the  saliva.  They  have  been  found  also  in  the 
liver,  spleen,  and  kidneys.  In  tertiary  lesions  they  appear  to  be 
much  less  numerous.  As  a  sequelae  to  the  tertiary  stage,  such 
conditions  as  general  paresis,  arteriosclerosis,  and  locomotor  ataxia 
frequently  result.  Recently  Noguchi  and  Moore  have  discovered 
the  spirochete  in  the  brain  of  a  certain  number  of  paralytic  insane 
cases. 

Immunity.  —  Immunity  in  syphilis  appears  to  be  somewhat 
different  to  that  produced  in  other  infectious  diseases.  All  at- 
tempts to  produce  active  immunity  artificially  have  so  far  failed, 
nor  is  passive  immunity  conferred  by  the  injection  of  serum  from 
an  animal  in  whom  the  disease  has  been  produced.  On  the  other 
hand,  man,  as  a  rule,  is  not  susceptible  to  reinfection  during  the 
active  stage  of  the  disease.  According  to  Colles's  law  a  mother 
who  gives  birth  to  a  syphilitic  infant  may  not  herself  contract 
the  disease,  but  may  develop  such  a  degree  of  immunity  that  she 
can  nurse  the  infant  without  becoming  infected  even  though  it 
has  venereal  ulcers  of  the  lips  and  tongue ;  whereas  the  child  would 
infect  the  healthiest  nurse  even  if  she  only  handled  and  dressed 
it.  The  converse  condition  is  stated  in  Profeta's  law ;  namely, 
an  infant  showing  no  taint  but  born  of  a  syphilitic  woman  may 
with  impunity  be  suckled  by  its  mother.  Exceptions  to  both  laws 
have,  however,  been  recorded. 

Microscopic  Examination.  —  Because  of  its  low  refractive  index 
T.  pallidum  is  best  seen  with  the  dark-stage  illumination.  Material 


262  BACTERIOLOGY   FOR  NURSES 

may  be  obtained  by  first  washing  the  lesion  with  sterile  water 
and  drying  it  with  sterile  gauze.  Part  of  the  base  of  the  ulcer 
is  then  scraped  with  a  curette  until  the  superficial  tissue  is  removed 
and  blood  appears.  The  blood  is  wiped  off  with  sterile  gauze  until 
clear  serum  begins  to  ooze.  A  drop  of  the  serum  mixed  with  a  drop 
of  distilled  water  is  placed  on  a  coverslip,  which  is  then  inverted 
over  a  hollow  slide  as  a  hanging  drop.  Examined  with  the  dark- 
stage  illumination  the  organisms  may  be  distinctly  seen  as  brightly 
illumined  objects  on  a  dark  background.  Films  may  also  be 
prepared  and  stained  as  already  described. 

Luetin.  —  Noguchi  has  prepared  an  extract  from  pure  cultures 
of  T.  pallidum  to  which  he  has  given  the  name  of  "  luetin,"  which 
gives  a  characteristic  reaction  in  syphilitic  individuals.  The 
reaction  is  analogous  to  the  tuberculosis  reaction  in  tuberculosis. 

Wassermann  Reaction.  —  The  complement  fixation  test  devised 
by  Wassermann,  Neisser,  and  Bruck,  whereby  the  presence  of 
specific  antibodies  in  serum  of  syphilitic  individuals  may  be  de- 
tected, is  described  in  Chapter  XIV.  The  test  is  widely  used  and 
gives  a  positive  reaction  in  over  90  per  cent  of  active  cases.  It  is 
practically  always  present  during  the  second  stage  and  tends  to 
disappear  as  the  disease  becomes  latent  or  is  cured. 


T.   PERTENUE 

A  spiral  organism  was  found  by  Castellani  in  1906  in  frambesia 
or  "  yaws,"  a  disease  occurring  in  tropical  countries.  The  lesions 
seem  to  be  analogous  to  those  of  syphilis,  and  by  some  writers  the 
diseases  are  considered  identical ;  other  observers  have  found 
that  the  antibodies  produced  against  the  two  organisms  differ, 
and  that  consequently  they  are  distinct  species,  and  yaws  cannot 
on  this  account  be  considered  as  a  mild  form  of  syphilis. 

S.   ICTEROHEMORRHAGI^ 

In  1915  Inada,  Ido,  and  other  Japanese  workers  demonstrated 
the  presence  of  a  spirochete,  to  which  they  gave  the  name  S.  ictero- 


SPTROCHETE   OBERMEIERI  263 

hemorrhagise  in  cases  of  infectious  jaundice  or  Weil's  disease. 
The  disease  is  characterized  by  irregular  fever,  often  severe  jaun- 
dice and  hemorrhagic  herpes.  The  organisms  appear  both  in 
the  blood  and  the  internal  organs.  Thus  the  disease  seems  to  be 
intermediate  between  the  two  classes  of  spirochetal  infections. 
The  relation  of  rats  to  the  spread  of  the  disease  has  been  estab- 
lished in  Japan  and  during  the  recent  war  in  the  trenches.  It 
has  been  found  that  the  proportion  of  infected  rats  is  sometimes 
as  high  as  30  per  cent;  the  spirochetes  are  passed  in  large  num- 
bers in  the  urine  of  infected  animals,  and  in  this  way  the  soil  and 
various  articles  become  contaminated. 


S.   OBERMEIERI   (S.   RECURRENTIS) 

Obermeier  discovered  in  1873  an  organism  in  the  blood  of 
patients  suffering  from  relapsing  fever  which  is  usually  known 
as  Spirocheta  obermeieri.  He  described  its  microscopical  appear- 
ance and  he  noted  its  presence  in  the  blood  during  the  time  of 
fever,  its  disappearance  about  the  time  of  the  crisis,  and  its  reap- 
pearance during  relapses. 

Morphology  and  Staining.  —  The  organisms  are  seen  as  long, 
delicate  filaments  from  16  to  40  /-t  in  length  and  about  0.5  /*  in 
width.  The  coils  are  somewhat  wide  and  irregular.  They  possess 
a  single  flagellum  at  one  end  and  move  in  a  partly  undulating, 
partly  twisting  fashion.  They  stain  faintly  with  the  anilin  dyes 
and  much  better  with  the  Romanowsky  stains.  They  are  Gram 
negative. 

Cultivation.  —  All  early  attempts  to  cultivate  the  organisms 
on  artificial  culture  media  were  unsuccessful.  One  investigator 
succeeded  in  keeping  them  alive  for  several  generations  by  placing 
them  in  celloidin  capsules  in  the  peritoneum  of  a  rat.  By  Nogu- 
chi's  method,  however,  they  can  be  readily  cultivated. 

Pathogenesis.  —  Rats,  mice,  and  monkeys  appear  to  be  the 
only  animals  susceptible  to  the  disease.  In  man  the  symptoms 
commence  with  severe  frontal  headache  and  a  rapid  rise  of  tem- 
perature, which  remains  high  for  five  to  seven  days  and  returns  to 


264  BACTERIOLOGY  FOR  NURSES 

normal  by  crisis.  About  a  week  later  a  relapse  occurs,  but  on  this 
occasion  the  fever  lasts  a  shorter  time  before  suddenly  disappear- 
ing; a  second  and  sometimes  a  third  relapse  occurs  after  about 
the  same  interval  of  time.  The  disease  is  usually  benign,  the 
mortality  varying  from  2  to  10  per  cent. 

Immunity.  —  Active  immunity  follows  recovery  from  an  attack 
of  the  disease,  and  the  blood  of  immunized  animals  will  confer  pas- 
sive immunity.  Metchnikoff  observed  that  during  the  fever 
the  spirochetes  were  rarely  taken  up  by  the  leukocytes  in  the  cir- 
culating blood,  but  that  at  the  time  of  the  crisis  the  organisms 
disappearing  from  the  blood  accumulated  in  the  spleen  and  were 
there  ingested  in  large  numbers  by  the  leukocytes.  These  observa- 
tions suggested  the  theory  that  the  immunity  produced  during  the 
first  period  of  fever  is  of  short  duration  and  does  not  last  until 
all  the  organisms  are  destroyed,  and  that  with  the  disappearance  of 
immunity  the  survivors  escape  from  the  internal  organs  and  appear 
again  in  the  blood  stream.  The  second  attack  is  less  severe  and 
is  of  shorter  duration  than  the  first,  and  with  each  succeeding  attack 
the  period  of  immunity  is  lengthened  until  finally  it  lasts  long 
enough  to  permit  all  the  organisms  to  be  killed. 

Varieties.  —  Several  distinct  diseases  are  caused  by  spirochetes 
similar  to  S.  recurrentis.  West  African  tick  fever  has  been  shown 
to  be  due  to  S.  duttoni,  a  spirochete  twice  as  long  as  S.  recur- 
rentis and  possessing  a  number  of  flagella.  East  African  tick 
fever  is  caused  by  a  third  variety,  S.  kochi,  and  relapsing  fever  as 
it  occurs  in  India  is  thought  to  be  caused  by  still  another  form. 
The  West  African  type  of  the  disease  was  shown  by  Dutton  to  be 
transmitted  by  a  species  of  tick.  Infected  insects  may  harbor 
the  parasites  for  several  months,  and  of  equal  importance  as  regards 
the  spread  of  the  disease  is  the  fact  that  the  spirochete  is  trans- 
mitted to  the  offspring  of  the  infected  tick  and  may  even  appear 
in  the  third  generation.  Strong  evidence  has  been  produced  for 
believing  that  head  and  body  lice  are  the  usual  agents  of  trans- 
mission of  European  relapsing  fever. 

Miscellaneous  Spirochetes.  —  In  addition  to  the  spirochetes 
causing  syphilis,  frambesia,  and  the  various  forms  of  relapsing 


MISCELLANEOUS   SPIROCHETES  265 

fever,  other  species  have  been  described  as  occurring  in  the  normal 
intestinal  tract  of  human  beings  and  mosquitoes,  in  various  gan- 
grenous processes  in  man,  and  in  the  blood  of  fowls  suffering  from 
a  disease  resembling  relapsing  fever.  Several  non-pathogenic 
forms  are  commonly  found  in  normal  mouths. 


CHAPTER  XXV 

PATHOGENIC   TRICHOMYCETES.     MOLDS.     YEASTS 

THE  trichobacteria  appear  to  hold  a  position  intermediate 
between  the  lower  forms  of  bacteria  and  the  molds.  Structurally 
and  functionally  they  are  more  complex  than  the  former  and 
much  simpler  than  the  latter.  Certain  authorities  consider  they 
should  all  be  grouped  together  under  the  name  of  streptothrix 
and  placed  with  the  molds  ;  other  authorities  hold  a  different  view. 

Hence   their    classification    is 
still  undecided. 

The  characteristics  of  the 
group  are  (1)  an  irregular, 
thread-like  growth  of  interde- 
pendent segments,  one  end  of 
which  may  be  free  while  the 
other  remains  attached  to  an 
object,  and  (2)  the  develop- 
ment of  a  special  portion  of 
the  organism  for  the  purpose 
of  reproduction  and  a  tendency 

FIG.  38.  —  Trichomycetes.  i  i  •  t  i       /TJ- 

to  branching  true  or  false  (r  ig. 

38).  In  false  branching  two  terminal  cells  are  developed  from  a 
parent  cell,  one  of  which  is  pushed  aside  but  remains  partly 
attached  to  the  main  stem.  As  the  two  cells  continue  to  develop 
the  appearance  of  branching  is  produced.  In  certain  forms,  when 
reproduction  is  about  to  take  place,  small,  rounded  cells  known  as 
conidia  or  spores  appear  either  at  the  free  end  of  the  organism  or 
at  intervals  along  the  filament,  from  which  new  individuals  de- 
velop. Such  spores  are  more  closely  related  to  those  of  the  molds 

266 


PATHOGENIC   TRICHOMYCETES  267 

and  should  be  differentiated  from  the  spores  of  the  lower  bacterial 
forms  since  they  do  not  possess  the  same  high  resistance  and  serve 
only  to  reproduce  the  species.  The  forms  known  to  be  pathogenic 
for  man  may  be  arranged  in  four  main  groups : 

1.  Leptothrix.      Almost   straight   thread-like  growth.     No 

branching. 

2.  Cladothrix.     False  branching. 

3.  Nocardia.     True  branching.     Reproductive  elements. 

4.  Actinomyces.     True   branching.     Characteristic    wreath- 

like  growth  in  tissues. 

Leptothrix.  —  Suppurative  conditions  of  the  mouth  have  been 
reported  as  due  to  a  member  of  this  class.  Investigators  have 
considered  that  Leptothrix  buccalis,  a  form  frequently  found  in 
normal  mouths,  may  under  certain  conditions  become  pathogenic. 
Since  the  organisms  have  been  so  little  studied,  however,  con- 
firmatory evidence  has  not  yet  been  obtained. 

Cladothrix.  —  Several  infections  have  been  reported  as  due  to 
this  group,  but  some  difficulty  has  been  experienced  in  deciding 
whether  the  organisms  should  be  classed  as  cladothrix  or  nocardia, 
since  the  only  morphological  difference  existing  between  the  two 
forms  is  that  of  true  or  false  branching.  An  organism  isolated  at 
autopsy  by  Eppinger  from  a  chronic  cerebral  abscess  which  had 
resulted  in  purulent  meningitis  was  considered  to  show  false  branch- 
ing and  given  the  name  cladothrix  asteroides.  On  artificial  media 
it  developed  a  delicate  fungoid  growth,  and  in  rabbits  and  guinea 
pigs  it  produced  an  infection  similar  to  that  observed  in  a  number 
of  lung  infections  usually  designated  "  pseudotuberculosis." 

Nocardia.  —  The  group  is  perhaps  more  frequently  named 
streptothrix,  although  according  to  the  rules  of  nomenclature 
the  name  is  not  applicable  since  it  was  given  as  early  as  1839  to  a 
species  of  mold.  Trevisan  in  1889  suggested  the  name  nocardia 
in  its  place  for  the  organism  discovered  by  Nocard  in  farcin  des 
boeufs.  Nocardia  have  been  found  in  brain  abscesses,  meningitis, 
wound  infections,  and  pneumonic  conditions.  Consolidated  areas 
in  the  lungs  and  nodular  formations  have  been  found  which  clini- 


268  BACTERIOLOGY  FOR  NURSES 

cally  so  closely  resembled  tuberculosis  that  no  means  of  distin- 
guishing the  two  forms  of  disease  could  be  devised  save  that  of 
finding  the  causative  organism. 

Subcutaneous  injections  into  rabbits  cause  the  formation  of 
abscesses,  which  when  incised  are  found  to  contain  a  thick  muci- 
laginous fluid;  intravenous  injections  produce  a  rapidly  fatal 
infection. 

In  smears  made  from  cultures  the  organisms  have  a  fine  slender 
appearance,  —  the  branching  is  unsymmetrical  and  almost  at 
right  angles  to  the  stem.  When  properly  stained  a  distinct  bead- 
ing of  the  protoplasm  may  be  observed;  stained  by  Gram's 
method  they  retain  the  violet  color.  In  broth  cultures  growth 
appears  as  minute  white  fluffy  tufts  clinging  to  the  sides  of  the 
tube  when  left  undisturbed.  Small  colonies  appear  on  Loeffler's 
serum  after  three  to  five  days'  incubation  at  37°  C. 

Actinomyces.  —  Actinomycosis  has  been  the  most  studied  of 
the  group  of  diseases  caused  by  the  higher  bacteria.  It  occurs 

chiefly  in  cattle,  but  occasionally  in 
other  animals  and  in  man.  The  dis- 
ease was  described  early  in  the  nine- 
teenth century  under  the  name  of 
osteosarcoma.  Later  in  1877  Bollinger 
discovered  the  specific  parasite,  and 
the  botanist  Harz,  who  studied  the 
organism,  gave  to  it  the  name  Actino- 
myces or  ray  fungus,  on  account  of 

FIG.  39.— Actinomyces.  'he  ™y-like  formation  of  its  growth 
in  the  tissues. 

Actinomycosis  is  an  inflammatory  condition  characterized 
by  the  presence  of  "  granules,"  which  are  small  round  masses  or 
colonies  of  the  parasite.  To  the  naked  eye  the  largest  appear 
as  yellow  or  greenish  points  about  the  size  of  a  small  pin's  head. 
When  suppuration  has  occurred  they  appear  free  in  the  pus,  other- 
wise they  may  be  found  embedded  in  the  granulation  tissue. 
According  to  their  age  and  their  structure  they  may  appear  as  a 
whitish  yellow,  a  green  black,  or  more  rarely  red.  Microscopically 


ACTINOMYCES  269 

each  granule  is  found  to  consist  of  one  or  many  rosettes  of  club- 
shaped  organisms  arranged  in  the  definite  radial  manner  which 
suggested  their  name.  Each  rosette  is  composed  of  threads  which 
radiate  from  a  center  and  terminate  in  glistening  club-like  endings 
closely  packed  together  (Fig.  39).  The  clubs  are  especially  found 
in  lesions  where  the  tissue  appears  to  be  displaying  a  degree  of 
resistance  to  the  growth  of  the  parasite.  Consequently  they  have 
been  thought  to  represent  a  means  of  defense  on  the  part  of  the 
parasites  against  the  action  of  the  phagocytes.  Other  observers 
have  considered  them  degenerative  portions  caused  by  contact 
with  the  body  fluids.  The  central  threads  show  true  branching 
and  in  the  older  colonies  a  tendency  to  segmentation  which  gives 
to  them  the  appearance  of  a  chain  of  cocci.  It  has  been  sug- 
gested that  the  coccus-like  bodies  may  be  spores  or  conidia.  The 
view,  however,  is  not  generally  held.  The  threads  stain  readily 
with  the  anilin  dyes  and  are  Gram  positive,  while  the  clubbed 
ends  lose  the  color  and  take  on  the  counter-stain. 

Cultivation.  —  The  organism  is  regarded  by  most  authorities 
as  a  strict  anaerobe.  On  agar  or  glycerin  agar  at  37°  C.  growth 
is  visible  after  several  days  as  small  yellowish  points  which  after 
becoming  confluent  resemble  somewhat  a  culture  of  tubercle  bacilli. 
The  organisms  penetrate  into  the  medium,  making  the  growth 
difficult  to  remove ;  in  broth  a  sediment  is  deposited  at  the  bottom 
of  the  tube  in  the  form  of  solid  white  granules.  In  order  to  obtain 
a  pure  culture  the  following  method  has  been  recommended: 
granules  are  removed  from  a  lesion,  thoroughly  washed  in  sterile 
water  to  remove  extraneous  organisms,  and  then  crushed  between 
two  sterile  coverslips.  A  microscopic  examination  is  made  to  be 
sure  a  filamentous  mass  is  present ;  otherwise,  especially  in  bovine 
material,  no  development  will  take  place.  If  filaments  are  seen 
a  portion  of  the  crushed  granule  is  transferred  with  a  platinum 
loop  to  tubes  of  melted  1  per  cent  glucose  agar  cooled  to  40°  C. 
and  thoroughly  distributed  through  the  medium.  At  the  same 
time  a  number  of  the  granules  after  washing  are  placed  on  the  side 
of  a  sterile  test  tube  and  allowed  to  dry  at  room  temperature  in 
the  dark.  In  this  way  all  contaminating  organisms  will  be  killed 


270  BACTERIOLOGY  FOR  NURSES 

by  drying,  and  should  first  cultures  be  unsuccessful  a  second  at- 
tempt may  be  made  by  using  the  dried  granules  in  the  same  manner 
as  the  fresh  ones.  If  after  several  days'  incubation  growth  has 
occurred  in  the  inoculated  tubes  colonies  appear  as  opaque  white 
nodules,  most  numerous  about  7  or  8  mm.  below  the  surface.  A 
characteristic  colony  may  be  cut  out  of  the  agar  by  means  of  a 
platinum  wire  and  transplanted  into  a  fresh  tube  of  melted  glucose 
agar. 

Resistance.  —  The  organisms  show  considerable  resistance 
to  drying.  On  the  walls  of  a  test  tube  they  may  be  found  alive 
after  seven  weeks  and  in  cultures  for  a  year  or  more. 

Pathogenesis.  —  Only  slight  local  lesions  can  be  produced  by 
the  inoculation  of  pure  cultures  into  the  smaller  animals  such  as 
guinea  pigs  and  rabbits ;  cattle,  however,  are  very  susceptible  to 
the  disease  and  in  a  less  degree  horses  and  swine  also.  In  cattle 
there  is  usually  an  abundant  growth  of  granulation  tissue  which 
results  in  large  tumor-like  masses.  The  disease  may  remain  local 
or  spread  by  continuity;  it  usually  appears  in  the  head  or  neck 
and  produces  the  condition  known  as  "  lumpy  jaw."  Lesions 
may  occur  in  the  lungs,  subcutaneous  tissue,  skin,  liver,  and  other 
organs.  Death  resulting  from  actinomycosis  is  due  rather  to  the 
mechanical  action  of  the  tumor  in  pressing  upon  or  occluding  the 
respiratory  or  alimentary  tract  rather  than  to  any  toxic  effect. 

In  man  the  disease  manifests  itself  in  a  similar  manner  save 
that  there  is  generally  less  production  of  new  tissue  and  more 
extensive  suppuration.  It  may  terminate  fatally  in  a  short  time 
through  a  secondary  infection  or  it  may  take  a  chronic  course 
for  years.  Treatment  with  potassium  iodide  has  effected  cures 
both  in  man  and  cattle  although  its  method  of  action  is  still 
unknown. 

Mode  of  Infection.  —  Transmission  by  direct  contact  has  not 
been  satisfactorily  proven.  Many  cases  in  human  beings  have 
been  reported,  in  which  so  far  as  could  be  discovered  no  contact 
with  a  previously  existing  case  had  occurred.  The  frequent  local- 
ization of  the  disease  in  the  head  and  jaw  has  led  to  the  supposi- 
tion that  the  organism  enters  the  body  by  way  of  the  mouth, 


HYPHOMYCETES  271 

probably  around  a  decayed  tooth,  or  the  crypts  of  the  tonsils,  or 
some  slight  abrasion.  Both  in  cattle  and  in  pigs  fragments  of 
grain  have  been  found  in  the  soft  tissues  of  the  mouth  embedded 
in  an  actinomycotic  growth,  and  since  there  is  a  certain  amount  of 
evidence  that  grain  is  the  natural  habitat  of  the  organism,  infection 
has  been  thought  to  occur  from  this  source.  Other  authorities 
maintain  that  the  parasite  is  present  in  normal  mouths  and  that 
penetration  depends  only  on  a  damaged  mucous  surface  and  a 
certain  degree  of  susceptibility  on  the  part  of  the  host. 

Mycetoma  (Madura  Foot) .  —  The  disease  resembles  actinomy- 
cosis  both  as  regards  the  character  of  the  lesions  and  the  occurrence 
of  the  parasite  in  the  form  of  granules.  They  are  nevertheless  un- 
doubtedly distinct.  Mycetoma  usually  appears  as  a  purulent 
inflammation  of  the  foot,  occasionally  of  the  hand  or  other  part 
of  the  body.  A  small  swelling  first  appears  which  gradually 
enlarges,  and  in  the  center  of  the  new  tissue  there  occurs  a  purulent 
softening  followed  by  ulceration.  Enlargement  and  distortion 
of  the  affected  part  and  frequently  necrosis  of  the  bones  occur. 
Within  the  softened  tissue  the  small  granular  bodies  may  be  seen, 
yellowish  pink  in  color  or  almost  black  like  grains  of  gunpowder. 
It  is  thought  by  some  observers  that  the  yellow  form  is  actinomy- 
cosis  and  that  the  black  variety  is  caused  by  a  member  of  the  hy- 
phomycetes  group.  Clinically  the  two  forms  of  the  disease  are 
identical. 

HYPHOMYCETES 

The  molds  and  trichobacteria  closely  resemble  each  other  in 
that  both  have  a  branching,  thread-like  growth.  The  life  history 
of  the  former,  however,  is  much  more  complicated  than  that  of 
the  latter. 

The  growth  of  the  hyphomycetes  is  characterized  by  a  mass 
of  tubular,  branched  filaments  termed  hypha,  which  interlace  one 
within  the  other,  forming  a  more  or  less  web-like  structure  known 
as  the  mycelium.  In  the  lower  forms,  the  phycomycetes,  each 
hypha  is  a  single  sometimes  branched  cell  except  when  reproductive 
organs  occur,  whereas  in  the  higher  forms,  the  my  corny  cetes,  the 


272  BACTERIOLOGY  FOR  NURSES 

hyphae  are  segmented  by  transverse  walls,  each  filament  consist- 
ing of  cells  placed  end  to  end. 

Among  the  phycomycetes,  of  which  Mucor  mucedo,  the  white 
cottony  mold  which  grows  in  damp  bread,  is  a  familiar  example, 
reproduction  may  take  place  asexually  or  sexually;  the  former 
method  is  the  most  usual.  The  end  of  a  hypha  becomes  shut  off 
by  a  transverse  wall  and  the  extremity  then  swells  into  a  globular 
sac  or  sporangium  within  which  numerous  oval  spores  develop. 
The  swelling  of  the  gelatinous  mass  in  which  the  spores  are  em- 
bedded ruptures  the  thin  cell  wall  and  the  spores  thus  escape. 

Other  forms  of  asexual  repro- 
duction occur  amongst  these 
lower  forms,  one  of  which  is 
the  production  of  thick-walled 
spores  termed  chlamydospores, 
which  possess  a  much  higher 
degree  of  resistance  than  the 
thin-walled  variety.  Under  cer- 
tain conditions  the  conjugation 
of  two  cells  precedes  spore  for- 
mation (sexual  reproduction). 
So-called  gametophores  occur 

FIG.  40.  —  Mucor  Mucedo.  ,1        •  •   -U-L 

as  outgrowths  m  neighboring 

hyphse,  and  when  the  tips  of  the  two  gametophores  come  in  con- 
tact they  fuse,  transverse  septa  are  formed,  and  a  zygospore  is 
the  result.  From  the  matured  zygospore  a  germ  tube  arises  which 
may  begin  to  function  at  once  in  the  usual  manner  (Fig.  40). 

The  mycomycetes  reproduce  almost  invariably  by  asexual  spore 
formation.  Two  main  groups  are  recognized  on  the  basis  of  their 
method  of  forming  spores.  In  one  series  a  cell  or  ascus  is  formed 
at  the  end  of  a  hypha  and  within  it  is  produced  a  number  of  spores 
constant  to  the  species.  The  number  is  always  a  multiple  of  two, 
usually  eight.  The  group  having  asci  is  known  as  ascomycetes. 
In  the  second  series  no  spore  sac  is  formed.  The  terminal  portion 
of  a  hypha  segments  into  germinating  branches  known  as  conidio- 
phores.  These  conidiophores  divide  into  two  or  three  branches, 


MOLDS 


273 


the  sterigmata.  From  these  other  sterigmata  may  be  produced, 
at  the  end  of  which  a  single  chain  of  constricted,  bead-like  spores 
or  conidia  are  formed.  Penicillium,  the  common  blue  mold,  is  a 
familiar  example  of  this  group  (Fig.  41). 

Molds  have  claimed  attention  probably  more  because  of  their 
ability  to  spoil  fruit  preserves  and  other  food  substances  than 
their  tendency  to  produce  disease.  Their  spores  are  practically 
ubiquitous  and  are  more  numerous  in  ordinary  air  than  bacteria. 
Mold  infection  of  plants,  such  for  example  as  potato  rot,  often  re- 
sults in  serious  economic  loss ;  other  infected  plants  may  if  ingested 
have  a  disastrous  effect  upon 
the  body  cells.  The  mold 
Claviceps  purpurea  which  in- 
fects rye  and  other  grain  has 
been  found  to  cause  a  condi- 
tion of  poisoning  known  as 
ergotism. 

Fortunately,  comparatively 
few  varieties  are  pathogenic  for 
man.  Pigeons  are  extremely 
susceptible  to  the  genus  Asper- 
gillus,  which  gives  rise  to  a 
form  of  pseudotuberculosis.  A 
number  of  cases  of  the  disease  have  been  reported  in  human 
beings,  especially  amongst  bird  fanciers.  A  case  of  mold  infection 
has  been  reported  which  at  autopsy  showed  multiple  abscesses  in 
the  brain,  lungs,  intestines,  and  peritoneum,  and  in  all  the  lesions 
a  species  of  mucor  was  found.  Eye  and  ear  infections  have  also 
been  attributed  to  the  same  organisms. 

Ringworm.  —  Ringworm  is  probably  the  most  frequently  met 
with  of  the  diseases  due  to  hypomycetic  growth.  It  is  contagious 
in  that  it  may  be  communicated  from  one  individual  to  another 
or  it  may  be  contracted  from  domestic  animals.  The  disease  is 
caused  by  at  least  two  different  members  of  the  species  Tricho- 
phyta  of  the  fungi  imperfecti  group,  Tinea  circinata  affecting  the 
body  and  Tinea  tonsurans  the  head.  The  fungi  are  more  parasitic 


FIG.  41. —  Penicillium. 


274  BACTERIOLOGY  FOR  NURSES 

than  certain  other  forms  in  that  they  penetrate  to  the  underlying 
tissue  instead  of  vegetating  on  the  surface  layer  of  the  skin.  The 
infection  probably  commences  first  in  the  epidermis  surrounding 
the  hair  bulb  and  from  thence  it  spreads  into  the  bulb  and  up 
into  the  hair  substance.  The  organisms  may  be  readily  seen  by 
removing  a  hair  with  the  bulb  attached  and  placing  it  in  a  drop  of 
sodium  or  potassium  hydroxide  solution  under  a  coverslip.  Ex- 
amined with  the  microscope,  enormous  numbers  of  interlaced 
threads  and  spores  may  be  seen  lying  within  the  bulb. 

Favus.  —  The  disease  is  contagious  and  is  produced  by  Achorion 
schoenleinii,  a  mold  discovered  by  Schoenlein  in  1839.  The  organ- 
ism grows  more  slowly  than  that  producing  ringworm.  It  affects 
both  the  hairy  and  smooth  parts  of  the  body  and  attacks  most 
frequently  the  skin  of  persons  whose  vitality  is  low.  Usually 
the  disease  appears  on  the  scalp,  but  may  attack  any  portion  of 
the  skin  or  even  the  mucous  membranes.  Growth  appears  as 
tiny  round  sulphur-yellow  disks  with  a  cup-like  depression  pierced 
in  the  center  by  a  hair.  The  spores  penetrating  into  the  hair 
follicles  cause  by  the  density  of  their  growth  such  pressure  upon 
the  tissue  beneath  that  the  vitality  of  the  hair  is  impaired.  Fre- 
quently the  hair  becomes  invaded,  the  shaft  especially  being 
affected.  The  chief  feature  of  the  disease,  however,  is  the  destruc- 
tion of  the  epithelial  cells  of  the  hair  follicles,  resulting  when  re- 
covery takes  place  in  the  formation  of  cicatricial  tissue. 

Pityriasis  Versicolor.  —  The  organism  giving  rise  to  this  condi- 
tion was  discovered  by  Eichstedt  in  1846  and  later  named  Micro- 
sporon  furfur.  It  appears  to  be  less  parasitic  than  the  fungus  of 
ringworm  and  flavus  and  attacks  only  the  superficial  layer  of  the 
skin.  The  growth  appears  as  a  scaly  eruption,  varying  in  color 
from  a  creamy  yellow  to  a  reddish  brown.  It  occurs  chiefly  in 
persons  living  under  uncleanly  conditions  or  in  those  who  have  a 
tendency  to  profuse  perspiration.  Several  of  the  lower  animals 
are  susceptible,  especially  the  cat. 

Sporotrichosis.  —  The  disease,  which  is  characterized  by  a  swell- 
ing of  the  lymphatics  and  a  chronic  ulcerative  condition,  was  shown 
by  Schenk  in  1898  to  be  due  to  another  member  of  the  fungi 


YEASTS  275 

imperfecti  group.  The  initial  infection  usually  takes  place  through 
some  slight  abrasion  of  the  skin  and  from  thence  spreads  along 
the  line  of  the  lymphatics.  Lesions  have  been  recorded  in  the 
larynx,  pharynx,  and  muscle  and  bone  tissues.  Sporotrichosis 
appears  in  the  past  to  have  been  frequently  confused  with  syphilis 
because  the  condition  readily  yields  to  treatment  with  iodin  com- 
pounds ;  mercury  salts  have  no  curative  effect. 

Thrush.  —  Infection,  which  is  most  frequent  in  young  infants, 
occurs  as  white  patches  of  fungoid  growth  on  the  tongue  and  fauces 
and  may  extend  into  the  esophagus.  General  infection  has  been 
reported,  with  abscess  formation  in  the  various  internal  organs. 
In  lesions  and  in  cultures  the  fungus  shows  characteristics  both 
of  the  molds  and  yeasts,  and  it  is  probable  that  it  occupies  a  posi- 
tion intermediate  between  the  two  forms.  The  yeast-like  por- 
tions are  oval  in  form,  averaging  about  5.5  p  long  and  4  /*  in 
diameter,  while  the  thread  formations  vary  greatly  in  length  and 
thickness.  Several  varieties  of  thrush  fungus  are  thought  to 
exist,  although  their  classification  at  present  is  by  no  means  clear. 
The  name  monilia  Candida  has  been  suggested  for  the  group.  The 
term  in  common  usage  is  Oidium  albicans. 


BLASTOMYCETES 

Yeasts,  like  molds,  have  been  studied  in  the  past  mainly  because 
of  their  economic  importance;  for  many  centuries  their  role  in 
the  brewing  and  baking  industries  has  been  recognized.  The 
main  characteristics  of  the  yeasts  is  their  mode  of  reproduction 
by  budding,  hence  their  name  of  blastomycetes  or  "  budding 
fungi  "  in  contrast  to  that  of  molds,  hyphomycetes,  or  "  thread 
fungi."  Nevertheless  no  strict  separating  line  can  be  drawn 
between  the  two  groups  since,  as  already  stated,  forms  exist  which 
possess  characteristics  of  both  groups. 

Ordinarily  yeast  cells  are  oval  in  shape,  each  cell  possessing 
a  more  or  less  definite  nucleus  and  surrounding  wall  of  cellulose ; 
they  vary  in  size  from  1  /*  in  diameter  in  old  cultures  to  giant 
cells  which  may  have  a  width  of  40  /*.  During  the  process  of 


276 


BACTERIOLOGY  FOR  NURSES 


FIG.  42.  —  Yeast  Cells. 


budding  the  nucleus  moves  to  the  edge  of  the  cell  and  commences 
to  divide.  Soon  a  protrusion  appears  which  rapidly  develops  into 
a  daughter  cell  of  the  same  shape  and  size  as  the  mother  cell. 
Under  certain  conditions  many  yeasts  are  able  to  reproduce  by 
spore  formation ;  the  nucleus  divides  usually  into  four  portions, 

each  of  which  becomes  the  cen- 
ter of  a  new  cell  lying  within 
the  parent  cell  (Fig.  42). 

Pathogenic  Yeasts.  —  Busse 
in  1894  was  the  first  to  report 
the  pathogenicity  of  a  yeast 
to  which  was  given  the  name 
Saccharomyces  busse.  In  the 
case  he  studied  the  first  lesion 
appeared  in  the  form  of  an 
abscess  on  the  tibial  bone. 
Thirteen  months  later  the  pa- 
tient died  from  a  generalized 
yeast  infection,  and  on  autopsy  the  yeast  was  found  in  lesions  in 
the  ulna,  lung,  kidney,  and  spleen. 

Since  the  discovery  by  Busse  many  similar  infections  have 
been  reported.  In  most  cases  a  small  papule  first  appears  with  a 
moderate  indurated  area  surrounding  it;  later  a  pustule  forms 
which  discharges  yellowish  pus.  The  lesion  spreads  slowly,  and 
as  it  invades  fresh  tissue  the  older  areas  show  a  tendency  to  heal. 
The  organisms  may  be  readily  demonstrated  in  film  preparations 
made  from  pus  in  the  usual  manner  and  stained  with  methylene 
blue.  For  their  cultivation  glucose  agar  is  the  most  suitable 
medium.  They  are  isolated  with  difficulty  from  material  in  which 
bacteria  are  growing  because  they  develop  more  slowly  than  the 
latter.  Repeated  plating  and  the  use  of  high  dilutions  is  generally 
necessary  for  their  isolation. 

The  tumor-like  growths  in  some  forms  of  blastomycosis  has 
led  certain  observers  to  assume  a  relationship  between  these  or- 
ganisms and  cancerous  growths.  The  assumption  has  not  yet 
been  supported  by  satisfactory  evidence. 


CHAPTER  XXVI 

THE  PATHOGENIC  PROTOZOA.  AMEB.E.  FLAGELLATA 

THE  lowest  forms  in  the  animal  kingdom,  the  protozoa,  are 
characterized  by  the  simplicity  of  their  structure  as  compared 
with  the  higher  animals,  the  metozoa.  For  the  most  part  each 
organism  consists  of  a  single  cell  composed  of  cytoplasm  and  nuclear 
substance.  Nevertheless,  although  unicellular  and  of  such  simple 
morphology  the  protozoa  are  much  more  complete  than  bacteria 
both  in  form  and  in  their  life  cycle. 

Morphology.  —  The  cytoplasm  of  the  cell  consists  usually  of 
an  outer,  dense  portion,  the  ectoplasm,  and  an  inner,  more  fluid 
portion,  the  endoplasm,  which  surrounds  one  or  more  nuclei  as  well 
as  various  granules  and  vacuoles.  Certain  of  the  latter  appear 
to  act  as  digestive  organs ;  others  show  periodic  contractile  move- 
ments and  serve  to  eject  waste  products  from  the  cell  body. 

In  many  of  the  protozoa  the  chromatin  substance  of  the  nucleus 
is  massed  together  in  a  deeply  staining  round  body  called  the 
karyosome,  and  embedded  in  the  karyosome  is  the  centrosome,  a 
small  body  always  present  in  metazoon  cells,  which  plays  an  im- 
portant part  in  cell  division.  In  certain  forms  still  another  definite 
portion  of  the  nuclear  chromatin,  the  kinetic  nucleus,  may  form 
the  root  of  a  flagellum.  The  kinetic  nucleus  may  be  distinct  or  it 
may  merge  into  another  small  body,  the  blepharoplast.  Each 
of  these  four  bodies,  the  karyosome,  centrosome,  kinetic  nucleus, 
and  blepharoplast,  have  their  origin  in  the  nucleus  of  the  cell. 

When  conditions  become  unsuitable  the  organisms  may  pro- 
tect themselves  by  forming  a  highly  resistant  enveloping  mem- 
brane. Such  an  encysted  form  will  withstand  extremes  of  heat  and 
cold  and  long  periods  of  drying,  and  then  as  soon  as  conditions 

277 


278  BACTERIOLOGY  FOR  NURSES 

become  again  suitable  the  organism  absorbs  water,  the  cyst  wall 
ruptures,  and  ordinary  development  is  recommenced. 

Nutrition  and  Reproduction.  —  Many  of  the  protozoa  obtain 
their  nourishment  by  the  absorption  of  the  fluid  food  directly 
through  the  cell  wall ;  others  are  able  to  ingest  solid  particles, 
such  as  bacteria,  through  a  suctorial  tube  or,  as  amongst  the  ameba, 
by  extending  a  portion  of  their  protoplasm  and  completely  sur- 
rounding the  food  morsel.  After  the  food  is  digested  the  waste 
substance  may  be  excreted  by  osmosis  or  by  means  of  a  contractile 
vacuole. 

The  simplest  form  of  reproduction  is  by  transverse  or  longitudinal 
division.  A  more  complex  form,  spoken  of  as  multiplicative 
reproduction,  or  brood  formation,  occurs,  in  which  the  nucleus 
divides  into  several  portions,  each  of  which  becomes  surrounded 
with  protoplasm  and  finally  separates  into  as  many  daughter 
cells.  Multiplicative  reproduction  without  conjugation  is  spoken 
of  as  schizogony  and  the  daughter  cells  are  known  as  merozoites ; 
when  after  fertilization  such  division  takes  place  within  a  cyst 
it  is  spoken  of  as  sporogeny  and  the  resulting  cells  are  called 
sporozoites. 

Many  forms  multiply  both  sexually  and  asexually,  some  of 
which  pass  the  sexual  phase  of  their  existence  in  one  host  and  the 
asexual  phase  in  another. 

The  forms  already  studied  that  are  pathogenic  for  man  are 
included  in  four  main  classes : 

1.  Rhizopodia.     Movement  by  means  of  temporary  protrud- 

ing portions  known  as  pseudopodia;  reproduction 
by  simple  division  or  multiplication  within  a  cyst; 
possession  of  one  or  more  nuclei.  Genus  parasitic  for 
man.  Entameba. 

2.  Flagellata.     Movement  by  means  of  flagella;    possession 

by  certain  forms  of  nucleus,  contractile  vacuoles,  and  a 
small  opening  for  food.  Genera  parasitic  for  man. 
Trypanosoma,  Leishmania. 

3.  Sporozoa.     May   form   pseudopodia;    food   ingested   by 

osmosis;    one  or  more  nuclei;    no  contractile  vacuole; 


THE   PATHOGENIC   PROTOZOA 


279 


reproduction  by  spores.     Genera  pathogenic  for  man. 
Coccidia,  Sarcosporidia,  Nosema,  Babesia,  Plasmodia. 
4.   Ciliata.     Movement  by  means  of  cilia;    reproduction  by 
transverse  division.    Germs  parasitic  for  man.    Balanti- 
dium. 

AMEB.ffi 

The  amebae  are  characterized  by  their  ability  to  project  por- 
tions of  their  protoplasm  into  pseudopodia,  or  "  false  feet  "  which 
serve  as  organs  of  locomotion 
and  nutrition.  The  pseudo- 
podia  may  protrude  from  any 
portion  of  the  cell  or  from  dif- 
ferent parts  at  the  same  time ; 
they  are  quite  irregular  in 
form  and  are  called  forth  only 
in  response  to  some  physical 
or  chemical  stimulus.  When 
such  a  stimulating  object  may 
be  used  as  food  the  pseudo- 
podia  flow  around  it  and 
eventually  absorb  it  into  the 
cell  protoplasm.  All  forms  of  ameba  possess  one  or  more  nuclei 
and  usually  a  contractile  vacuole.  Multiplication  takes  place  by 
simple  division  or  by  encysted  brood  formation  (Fig.  43). 

Saprophytic  forms  are  abundant  in  nature ;  they  may  be  found 
wherever  moisture  and  decaying  vegetable  matter  exist.  Yet,  not- 
withstanding their  common  occurrence,  little  is  known  of  their 
life  history.  Not  all  forms  of  protozoa  showing  ameboid  move- 
ment can  be  classified  as  rhizopodia  until  most  of  their  life  history 
is  known,  since  members  of  other  classes  may  pass  through  an 
ameboid  stage.  Some  flagellates,  for  example,  during  one  period 
of  their  existence  develop  blunt  pseudopods  and  crawl  along  as 
typical  ameba. 

Three  forms  of  ameba  have  been  described  as  parasitic  in  man : 
Entameba  histolytica,  E.  coli,  and  E.  gingivalis. 


FIG.  43.  —  Ameba. 


280  BACTERIOLOGY   FOR  NURSES 

E.  Histolytica.  —  As  early  as  1860  Lambl  of  Prague  discovered 
amebae  in  the  stools  of  a  severe  case  of  dysentery.  Very  soon  other 
investigators  reported  their  presence  both  in  dysenteric  and  in 
normal  stools,  with  the  result  that  a  number  of  parasitic  forms  were 
thought  to  exist.  Schaudinn  in  1903  clearly  showed  that  many 
of  the  forms  described  represented  different  stages  in  the  develop- 
ment of  one  organism  and  that  practically  only  two  intestinal 
forms  had  been  discovered,  which  he  renamed  E.  histolytica  and 
E.  coli.  The  latter  he  regarded  as  a  harmless  parasite  and  the 
former  as  the  inciter  of  amebic  dysentery. 

Amebic  dysentery  differs  from  bacillary  dysentery  in  that  it 
is  a  chronic  infection  of  the  colon  which  starts  insidiously  and 
is  characterized  by  relapses  and  recurrences.  It  occurs  sporadi- 
cally or  in  endemic  form  in  the  tropics  and  not  unfrequently  in 
the  temperate  zones  and  in  about  20  per  cent  of  cases  is  compli- 
cated by  liver  abscesses.  Emetin  administered  hypodermically 
has  proved  of  such  therapeutic  value  that  it  is  accepted  as  a  spe- 
cific. Bacillary  dysentery,  on  the  other  hand,  is  an  infection  of  the 
small  intestine,  has  an  acute  onset,  marked  symptoms  of  toxemia, 
occurs  in  epidemic  form,  usually  has  no  sequelae,  and  is  not  influ- 
enced by  emetin. 

Amebic  dysentery  runs  an  irregular  course  over  a  period  of  a 
few  weeks  to  several  years.  In  severe  forms  the  stools  are  watery 
and  contain  varying  amounts  of  blood  and  mucus ;  they  vary  in 
number  from  twenty  to  fifty  in  twenty-four  hours.  The  amebae 
penetrate  between  the  epithelial  cells  to  the  submucosa,  multiply 
there,  and  by  their  presence  irritate  the  tissues.  At  first  there 
is  an  edematous  local  swelling ;  soon  the  mucous  membrane  becomes 
ulcerated,  and  gangrenous  sloughs  result.  The  ulcers  thus  formed 
have  an  irregular  overhanging  border  with  a  much  larger  cavity 
in  the  submucosa  than  the  opening  into  the  mucous  membrane 
indicates. 

When  liver  abscesses  occur  as  a  complication  they  are  usually 
single  and  of  a  large  size,  or  occasionally  numerous  small  ones 
may  be  seen.  The  contents  usually  consist  of  a  gelatinous  pink 
fluid  containing  necrosed  tissue,  blood,  and  amebae.  It  not 


THE  PATHOGENIC  PROTOZOA  281 

infrequently  happens   that  such  abscesses  rupture  through  the 
diaphragm  into  the  lungs  or  into  the  peritoneal  cavity. 

E.  histolytica  has  been  shown  to  be  pathogenic  for  cats,  dogs, 
and  monkeys,  provided  infection  takes  place  either  by  feeding 
them  with  material  containing  cysts  or  by  rectal  inoculation  of 
vegetative  forms. 

The  source  of  pathogenic  amebic  infection  is  not  definitely 
known.  In  Manila  the  water  supply  is  considered  responsible  for 
its  transmission.  Recent  experiments  on  Filipinos  who  acted  as 
volunteers  tend  to  show  that  E.  histolytica  is  a  strict  parasite, 
and  consequently  infection  can  only  come  from  an  individual 
harboring  the  organism  in  the  intestines.  Since  the  organism 
always  enters  the  body  by  the  mouth  and  leaves  in  the  feces,  trans- 
mission evidently  occurs  through  the  ingestion  of  substances 
contaminated  with  infected  excretions. 

Examination  of  Feces.  —  In  fresh  specimens  E.  histolytica  ap- 
pears as  a  large,  round  or  oval  body  20  to  30  /x  in  diameter ;  the 
nucleus  is  pale  and  not  readily  seen.  Usually  there  are  numerous 
digestive  vacuoles  in  which  red  blood  cells  may  be  seen.  Often 
in  unstained  preparations  the  amebse  appear  to  be  of  a  greenish 
color,  due,  it  is  thought,  to  the  hemoglobin  liberated  from  the 
ingested  red  cells.  When  in  the  cyst  form  they  are  small  and  round 
and  show  four  distinct  spherical  nuclei. 

The  feces  from  a  suspected  case  should  be  examined  as  soon 
as  possible  after  being  passed ;  a  loopful  of  the  slimy  portion  is 
diluted  with  physiological  salt  solution  and  examined  in  a  hanging 
drop,  preferably  on  a  warm  stage.  By  this  means  the  kind  of  move- 
ment and  the  nature  of  the  vacuoles  may  be  observed.  The  addi- 
tion of  a  drop  of  1  to  500  neutral  red  solution  will  stain  the  amebse 
a  pale  pink  color. 

Cultivation.  —  After  many  unsuccessful  attempts  to  cultivate 
amebse,  Musgrave  and  Clegg  devised  the  following  ingenious 
method.  Agar  medium  was  poured  into  sterile  Petri  dishes,  and 
after  hardening  several  rings  of  a  pure  culture  of  dead  bacteria 
were  spread  around  the  medium.  A  loopful  of  the  material  con- 
taining the  amebse  was  placed  in  the  center  of  the  dish.  It  was 


282  BACTERIOLOGY  FOR  NURSES 

found  that  as  the  amebse  multiplied  they  traveled  toward  the 
edge,  and  in  passing  the  rings  of  bacteria  they  deposited  the  living 
organisms  with  which  they  started  and  took  up  the  dead  ones. 
Thus  after  forty-eight  to  seventy-two  hours  active  amebse  free 
from  living  bacteria  were  found  at  the  periphery.  It  has  recently 
been  found  that  certain  strains  of  intestinal  amebse  will  grow  in 
pure  culture  when  inoculated  on  sterile  tissue  such  as  brain,  liver, 
or  kidney  placed  in  nutrient  agar. 

E.  Coli.  —  Various  investigators  have  reported  the  presence 
of  E.  coli  in  from  20  to  60  per  cent  of  all  normal  stools  examined 
regardless  of  locality.  The  organisms  vary  from  20  to  40  /*  in 
diameter.  As  a  rule  they  are  less  actively  motile  than  E.  histolyt- 
ica  and  contain  more  and  larger  vacuoles,  which  usually  are  filled 
with  bacteria  and  rarely  with  the  red  blood  cells  so  palatable  to 
the  latter.  Another  distinguishing  characteristic  is  their  division 
in  the  encysted  form.  E.  histolytica  usually  produces  only  four 
daughter  cells,  while  the  normal  number  for  E.  coli  is  eight.  Ex- 
periments have  demonstrated  that  parasitism  may  be  brought 
about  by  feeding  human  beings  with  E.  coli  cysts.  No  disease, 
however,  results,  though  the  amebse  may  be  present  in  the  intes- 
tines for  years. 

Entameba  Gingivalis.  —  The  organism  is  almost  invariably 
present  in  pyorrhea  alveolaris,  but  it  is  also  present  in  normal 
mouths,  and  as  yet  no  experimental  evidence  has  established  its 
relation  to  the  disease.  The  organism  can  readily  be  found  in 
tartar  scraped  from  the  teeth  near  the  gum  margin.  It  measures 
from  12  to  20  /*  in  diameter,  contains  a  nucleus  and  many  food 
vacuoles.  Multiplication  occurs  only  in  the  vegetative  stage  and 
then  by  simple  division.  Cyst  formation  does  occur,  but  it  is 
purely  a  protective  stage  and  not  one  of  reproduction. 

FLAGELLATA 

The  flagellata,  as  their  name  implies,  are  characterized  by  the 
possession  of  one  or  more  flagella.  They  are  divided  into  several 
subclasses.  Those  pathogenic  to  man  belong  chiefly  to  the  genera 
Trypanosoma  and  Leishmania. 


THE  PATHOGENIC  PROTOZOA  283 

Trypanosomes.  —  The  structure  of  the  trypanosomes  while 
varying  in  detail  is  more  or  less  uniform  for  the  entire  genus. 
The  body  is  long  and  flexible,  tapering  anteriorly  to  a  fine  point ; 
the  posterior  extremity  is  always  less  sharp  and  often  quite  blunt. 
The  nucleus  is  usually  situated  in  the  center  of  the  cell  and  behind 
it,  often  near  the  posterior  end,  is  the  kinetic  nucleus.  The  flagel- 
lum  rises  from  the  blepharoplast,  which  is  located  near  to  or  within 
the  kinetic  nucleus,  and  reaching  the  surface,  turns  forward  and 
forms  the  edge  of  a  thin  fluted  fold  of  ectoplasm,  the  undulating 
membrane,  which  runs  the  entire  length  of  the  cell,  and  then  con- 
tinues forward  as  a  thin,  thread-like  filament ;  a  smaller  flagellum 
occasionally  is  formed  which 
is  directed  backwards  and  acts 
as  a  rudder.  During  life  the 
constant  wave-like  motion  of 
the  undulating  membrane  and 
the  lashing  of  the  flagellum 
enables  the  organism  to  move 
with  great  rapidity. 

The  average  length  is  about 
30  p  and  the  width  1.5  to 
3  p.  Usually  one  to  several 
contractile  vacuoles  as  well  as 

food    vacuoles     may     be     Seen.  FIG.  44. -Trypanosomes. 

Occasionally  there  is  a  definite  grooved  opening  or  cytosome  for 
the  entrance  of  food.  Pseudopodia  may  develop  during  one  phase 
of  existence,  but  they  are  transitory. 

Multiplication  ordinarily  occurs  by  longitudinal  division ;  in 
certain  forms  reproduction  takes  place  within  a  cyst  after  ferti- 
lization. 

Cultivation  of  the  trypanosomes  outside  of  the  body  was  first 
accomplished  by  Novy  and  MacNeal  in  1903.  They  prepared 
a  medium  consisting  of  equal  parts  of  nutrient  agar  and  defibrinated 
rabbit's  blood.  On  such  medium  at  the  end  of  several  days  a  fairly 
good  growth  may  be  obtained. 

The  presence  of  the  organism  may  usually  be  demonstrated 


284  BACTERIOLOGY  FOR  NURSES 

in  a  hanging  drop  preparation  of  freshly  drawn  blood.  If  tryp- 
anosomes  are  present  attention  is  soon  attracted  to  their  neigh- 
borhood by  a  disturbance  amongst  the  red  blood  corpuscles,  and 
soon  the  rapidly  undulating  organisms  come  into  view.  Blood 
films  may  also  be  made  and  colored  with  Romanowsky  stain. 

With  the  exception  of  dourine,  a  disease  occurring  amongst 
horses,  the  trypanosomes  are  transmitted  from  one  animal  to  an- 
other through  the  agency  of  blood-sucking  insects  or  leeches.  The 
organism  undoubtedly  passes  through  a  definite  cycle  of  develop- 
ment within  its  invertebrate  host,  the  details  of  which,  however, 
are  not  yet  known. 

T.  Lewisi.  —  The  first  of  the  species  to  be  more  or  less  fully 
described  was  T.  lewisi,  a  comparatively  non-virulent  form  seen 
in  the  blood  of  the  rat  as  early  as  1845,  but  more  fully  studied  and 
described  by  Lewis  in  1879.  Since  then  it  has  been  noted  by  ob- 
servers in  various  parts  of  the  world. 

The  organism  is  peculiar  to  the  rat  and  of  no  importance  so 
far  as  human  infection  is  concerned,  but  in  that  animal  it  produces 
a  malady  which  runs  a  definite  course  although  it  is  rarely  fatal. 

When  a  rat  is  experimentally  infected  by  injecting  infective 
material  into  the  peritoneal  cavity  the  organisms  soon  appear 
in  the  blood,  and  So  rapid  is  their  multiplication  there  that  within 
a  few  days  they  seem  to  be  almost  as  numerous  as  the  red  blood 
cells.  The  infection  continues  for  about  two  months,  during 
which  time  the  animal  may  show  no  symptoms  of  disease.  At  the 
end  of  that  period  the  parasites  gradually  disappear  and  the  rat 
is  immune  against  further  infection.  The  serum  of  such  a  rat 
has  highly  protective  properties  and  a  marked  agglutinative  capac- 
ity, causing  the  trypanosomes  to  gather  together  in  a  rosette  for- 
mation in  which  the  flagella  are  directed  outward. 

The  rat  flea  is  responsible  for  the  transmission  of  the  parasite. 
It  becomes  infective  about  a  week  after  biting  a  diseased  animal 
and  remains  so  for  the  rest  of  its  life,  passing  the  trypanosomes 
in  its  dejecta.  The  organism  may  enter  through  the  puncture 
made  by  the  flea  in  the  act  of  sucking  or  may  be  swallowed  by 
the  rat  when  licking  its  fur. 


THE  PATHOGENIC  PROTOZOA  285 

T.  Evansi.  —  A  disease  of  horses  known  as  surra,  which  is  very 
prevalent  in  India,  was  shown  by  Evans  to  be  due  to  a  trypanosome. 
Experiments  have  shown  that  the  disease  is  transmitted  by  flies. 

T.  Brucei.  —  Nagana  or  tsetse  fly  disease  is  prevalent  in  South 
Africa  and  especially  in  Zululand,  where  it  affects  chiefly  horses, 
cattle,  and  dogs.  In  1894  Bruce  discovered  that  the  blood-in- 
fected animals  swarmed  with  the  trypanosomes  now  known  by 
his  name.  The  disease,  which  is  similar  to  the  surra  of  India,  is 
characterized  by  a  watery  discharge  from  the  eyes  and  nose,  swell- 
ings on  the  surface  of  the  abdomen  and  legs,  and  an  increasing 
emaciated  and  anemic  condition  which  generally  results  in  death 
after  several  weeks.  It  had  been  noticed  that  the  disease  was 
frequently  contracted  by  horses  passing  through  hot,  damp,  fly- 
infested  areas,  and  the  cause  was  thought  to  be  due  to  a  poison 
secreted  by  the  fly  and  transmitted  in  the  act  of  biting.  After 
a  number  of  experiments  Bruce  was  able  to  demonstrate  conclu- 
sively that  while  the  tsetse  fly  was  responsible  for  the  transmission 
of  the  causal  agent  the  latter  was  not  a  poisonous  secretion  but 
living  trypanosomes.  Bruce  found  that  the  organisms  are  more 
or  less  harmless  parasites  of  the  big  game  animals  of  South  Africa. 
Consequently  he  concluded  that  it  is  from  them  the  tsetse  flies 
derive  the  parasites  with  which  they  infect  domestic  animals. 

T.  Equiperdum.  —  A  trypanosomiasis  known  as  dourine  occurs 
among  horses  in  various  parts  of  Europe,  and  has  occasionally 
developed  in  some  of  the  northern  states  of  America  and  western 
Canada  amongst  imported  horses.  A  characteristic  of  the  disease 
is  that  so  far  as  is  known  it  is  transmitted  by  coitus  and  not  by 
biting  insects.  The  disease  is  usually  of  a  chronic  nature ;  the 
animal  becomes  paralyzed  and  death  occurs  as  a  rule  in  from  two 
to  ten  months. ' 

T.  Gambiense.  —  Within  comparatively  recent  years  the  im- 
portant discovery  was  made  that  the  terrible  disease  known  as 
sleeping  sickness,  which  affects  the  natives  of  South  Africa  and  also 
Europeans,  is  a  form  of  human  trypanosomiasis.  The  parasites 
found  by  the  first  investigators  are  grouped  together  under  the 
name  T.  gambiense.  They  closely  resemble  T.  equinum  and  T. 


286  BACTERIOLOGY  FOR  NURSES 

brucei  and  are  also  conveyed  by  the  bite  of  a  tsetse  fly,  Glossina 
palpalis,  belonging  to  the  same  genus  as  the  fly  responsible  for  the 
transmission  of  nagana. 

The  symptoms  of  the  disease  are  divided  into  two  stages. 
In  the  first  they  are  of  a  mild  character,  the  pulse  and  respirations 
are  quickened ;  there  is  an  irregular  elevation  of  temperature  and 
enlargement  of  the  lymph  nodes.  The  trypanosomes  are  found  in 
small  numbers  both  in  the  enlarged  glands  and  in  the  blood.  After 
several  months  and  in  some  cases  years  the  second  stage  of  the 
disease  commences.  The  fever  becomes  hectic,  the  patient  is  listless 
and  apathetic,  neuralgic  pains,  trembling  of  the  muscles,  and  grad- 
ually increasing  emaciation  and  lethargy  develop,  until  finally  a 
comatose  condition  occurs  and  death  ensues.  The  duration  of 
the  second  stage  is  from  four  to  eight  months,  during  which  period 
the  parasites  may  always  be  found  in  the  spinal  fluid.  So  far  as 
is  known  the  disease  is  invariably  fatal,  and  so  prevalent  is  it  in 
certain  parts  of  South  Africa  that  in  some  villages  from  30  to  50 
per  cent  of  the  inhabitants  have  been  found  to  be  infected. 

Treatment  with  atoxyl  and  arsenic  compound  is  said  to  have 
been  of  benefit  in  animal  trypanosomiasis.  In  human  cases,  how- 
ever, the  results  have  been  more  uncertain. 

T.  Rhodesiense.  —  Another  trypanosome,  T.  rhodesiense,  iso- 
lated in  1911  from  cases  of  sleeping  sickness  in  Rhodesia,  differs 
morphologically  from  T.  gambiense  and  is  also  more  virulent.  It 
is  conveyed  by  the  tsetse  fly  (Glossina  morsitans)  and  is  a  harm- 
less parasite  of  certain  wild  South  African  animals. 

Still  another  form  of  human  trypanosomiasis  transmitted  by  a 
bug  occurs  in  Brazil,  where  it  is  known  as  "  Chagas  disease." 


LEISHMANIA-DONOVANI 

Leishman  discovered  in  1900  in  the  blood  of  a  number  of  soldiers 
suffering  from  a  febrile  disease,  who  had  been  invalided  home  from 
Dum-Dum,  an  unhealthy  section  of  India,  peculiar  bodies  which 
he  thought  resembled  degenerating  forms  of  T.  brucei.  In  1903 
he  published  his  observations  and  suggested  that  the  cachexial 


THE  PATHOGENIC  PROTOZOA  287 

fever  so  prevalent  in  India  might  be  a  form  of  trypanosomiasis. 
Later  in  the  same  year  Donovan,  working  independently  in  India, 
confirmed  Leishman's  findings,  and  the  organisms  became  known 
as  the  Leishman-Donovan  bodies.  The  disease  to  which  they 
give  rise  has  been  called  by  a  variety  of  names  in  different  sections 
of  the  tropics,  e.g.  dumdum  fever,  non-malarial  cachexia,  and 
kala-azar.  Recent  investigators  have  reported  cases  in  many 
parts  of  India,  China,  Turkestan,  Algiers,  Egypt,  Italy,  and 
Greece. 

Kala-azar  is  characterized  by  fever  of  an  irregular  type,  a  pecul- 
iar dark  earthy  pallor  of  the  skin,  progressive  anemia  and  emacia- 
tion with  enlargement  of  the  spleen  and  liver,  and  frequently 
edematous  swellings  and  ulcers  of  the  skin.  The  disease  is  chronic, 
continuing  for  several  years  and  having  in  about  80  per  cent  of 
cases  a  fatal  ending. 

In  a  film  preparation  made  from  the  spleen  the  characteristic 
bodies  appear  round  or  oval  and  usually  2.5  to  3.5  /*  in  diameter. 
Within  the  protoplasm  two  intensely  stained  bodies  can  be  dis- 
tinguished :  one,  large,  round  or  heart-shaped,  situated  near  the 
periphery ;  the  other,  rod-shaped  and  generally  distinct  from  the 
larger  body.  The  organisms  may  be  seen  free  or  packed  within 
phagocytic  cells. 

In  the  body  the  parasite  multiplies  ordinarily  by  simple  division. 
Sometimes,  however,  multiplicative  reproduction  occurs,  the 
nucleus  dividing  several  times  within  the  cytoplasm  and  giving 
rise  to  a  corresponding  number  of  new  organisms. 

Cultivated  outside  of  the  body  on  Novy  and  MacNeal's  medium 
the  organism  develops  into  a  flagellate  form.  The  cell  lengthens  to 
about  20  fji.  The  smaller  nucleus  moves  near  to  one  end  and  from 
it  arises  a  long,  fine  flagellum.  The  whole  development  occupies 
about  ninety-six  hours.  It  was  not  until  the  organisms  were  culti- 
vated on  artificial  medium  that  their  relationship  to  the  flagellates 
was  established. 

Nothing  definite  is  known  as  to  the  manner  in  which  the  disease 
is  spread.  Bedbugs  fed  on  kala-azar  patients  have  been  found 
to  harbor  flagellates,  but  since  a  variety  of  similar  organisms 


288  BACTERIOLOGY  FOR  NURSES 

may  normally  inhabit  the  intestines  of  the  insects  the  evidence  is 
not  generally  accepted  as  conclusive. 

Leishmania  Infantum.  —  In  the  southern  parts  of  France, 
Portugal,  Greece,  and  Italy  a  disease  apparently  identical  with 
kala-azar  occurs,  affecting  children  between  two  and  five  years 
of  age.  The  fact  that  in  the  regions  in  which  it  occurs  the  infec- 
tion is  confined  to  young  children  has  given  rise  to  the  name 
Leishmania  infantum.  A  similar  disease  occurs  in  dogs  in  the  same 
regions,  and  the  view  has  been  advanced  that  the  infection  of 
children  may  be  through  the  agency  of  dog  fleas. 

Leishmania  Tropica.  —  In  the  tropics  and  also  in  subtropical 
regions  a  chronic  ulceration  of  the  skin  is  widely  prevalent  which 
is  known  by  various  names,  such  as  tropical  ulcer,  Delhi  sore, 
Aleppo  boil,  etc.  From  these  ulcers  bodies  have  been  obtained 
which  appear  to  be  identical  with  Leishmania-donovani.  The 
close  similarity  of  the  organisms  isolated  from  the  three  forms  of 
disease  suggests  that  they  may  be  of  the  same  species.  Knowledge 
of  their  relationship,  however,  is  as  yet  incomplete. 


CHAPTER  XXVII 

SPOROZOA.     CILIATA 

THE  sporozoa,  as  their  name  implies,  are  characterized  by  their 
method  of  reproduction  through  spore  formation.  They  are 
strict  parasites  and  are  for  the  most  part  harmless;  only  a  few 
species  are  known  to  be  pathogenic.  The  organisms  vary  greatly  in 
size,  in  structure,  and  in  development.  Certain  forms  are  so  small 
that  several  may  be  contained  within  a  single  red  blood  corpuscle, 
while  others  are  large  enough  to  be  seen  with  the  naked  eye.  Some 
members  of  the  group  show  ameboid  movements  during  certain 
phases  of  their  existence,  but  the  pseudopodia  serve  only  as  a  means 
of  locomotion  and  not  of  nutrition.  Their  life  history  is  more  or 
less  complicated,  one  period  being  passed  in  one  host  and  the  other 
period  within  the  body  of  another  species ;  or  different  phases  of 
development  may  take  place  in  different  tissues  of  a  single  host. 

The  known  pathogenic  forms  occur  among  five  different  genera : 
Coccidia,  Sarcosporidia,  Nosema,  Babesia,  and  Plasmodia. 

Coccidia.  —  A  disease  of  rabbits  occurs  in  epidemic  form  due 
to  Coccidium  cuniculi ;  a  few  cases  of  human  infection  have  been 
reported. 

Sarcosporidia.  —  Organisms  belonging  to  this  group  affect 
mainly  swine  and  cattle;  a  case  of  human  sarcosporidiosis  was 
reported  in  1909  in  Panama. 

Nosema.  —  So  far  as  is  known  no  infection  attributable  to  the 
nosema  has  occurred  in  man.  One  member,  however,  is  of  interest 
in  that  it  is  the  cause  of  pebrine,  the  infectious  disease  of  silkworms, 
to  which  Pasteur  devoted  several  years  of  study. 

Babesia    (Piroplasma).  —  Babes    was    the    first    to    observe 
the  parasites  in  the  blood  of  Roumanian  cattle.     Later,  in  1893, 
u  289 


290  BACTERIOLOGY  FOR  NURSES 

Theobald  Smith  described  them  more  fully  and  established  their 
relationship  to  the  disease  of  cattle  known  by  various  names,  such 
as  Texas  fever,  tick  fever,  hemoglobinuria,  and  red-water  fever. 
The  disease  is  characterized  especially  by  destruction  of  the  red 
blood  corpuscles  and  infection  of  the  spleen  and  liver.  So  far  as  is 
known  ticks  are  solely  responsible  for  its  spread.  After  fertilization 
the  female  gorges  herself  with  blood,  then  drops  to  the  ground 
and  there  lays  about  2000  eggs,  depositing  within  the  shell  of  each 
sufficient  blood  to  serve  the  embryo  as  food.  The  insect  dies 
within  a  few  days  after  the  egg  laying  has  been  completed.  The 
eggs  hatch  in  about  three  weeks,  and  the  larvae,  containing  within 
their  bodies  some  of  the  blood  of  their  mother,  crawl  about  until 
they  die  or  have  the  opportunity  of  attaching  themselves  to  another 
animal.  If  the  larvae  are  the  progeny  of  an  infected  mother  they 
thus  become  the  means  of  further  disseminating  the  disease. 

Similar  infections  affecting  other  animals  have  been  shown  to 
be  due  to  Babesia-like  parasites. 

Plasmodia.  —  For  many  centuries  malaria  has  appeared  in 
certain  regions  as  a  veritable  scourge,  yet  nothing  definite  was 
discovered  as  to  its  cause  until  the  latter  part  of  the  nineteenth 
century.  The  fact  that  it  remained  prevalent  in  certain  areas 
and  not  in  others  led  to  the  supposition  that  atmospheric  condi- 
tions were  in  some  way  responsible  and  to  its  being  termed  "  ma- 
laria "  or  "  bad  air." 

With  the  establishment  of  the  theory  that  each  infectious 
disease  is  caused  by  a  specific  infecting  organism  the  study  of 
malaria  was  taken  up  with  enthusiasm.  Several  investigators 
described  bacterial  forms  which  they  thought  might  be  the  causal 
agents,  but  their  findings  were  disproved.  In  1880  Laveran, 
a  French  military  surgeon  stationed  in  Algiers,  announced  that 
he  had  discovered  a  parasite  in  the  blood  of  malarial  patients 
which  was  found  to  be  a  protozoon  of  the  class  Sporozoa.  His 
discovery  was  confirmed  by  the  independent  researches  of  two 
Italian  investigators,  Marchiafava  and  Celli,  who  gave  the  organ- 
ism the  name  of  Plasmodiwn  malarias.  The  term  Hemameba  has 
been  suggested  as  more  appropriate  and  is  frequently  employed, 


PLASMODIA  291 

yet  according  to  the  rules  of  zoological  nomenclature  the  first 
given  name  must  be  retained. 

In  1896  Ross  found,  while  studying  a  form  of  malaria  affecting 
birds,  that  the  parasite  was  transmitted  from  infected  to  healthy 
birds  by  the  bite  of  the  common  mosquito,  Culex.  Reasoning 
from  this  fact,  he  concluded  that  the  malarial  parasite  giving  rise 
to  the  disease  in  human  beings  might  be  conveyed  in  a  similar 
manner.  After  a  long  series  of  experiments  he  found  that  the 
latter  could  not  develop  in  the  body  of  Culex,  but  that  it  did  develop 
in  another  variety,  Anopheles.  He  observed  rounded,  pigmented 
bodies  in  the  stomach  wall  of  Anopheles  that  had  been  fed  with 
the  blood  of  malarial  patients,  and  he  was  able  to  trace  all  the 
stages  of  development  from  the  time  the  organism  entered  the 
stomach  with  the  blood  until  it  settled  in  the  poison-salivary  gland 
of  the  insect  ready  to  be  transferred  again  to  a  human  host. 

Strikingly  conclusive  evidence  of  the  ability  of  mosquitoes 
to  transmit  infection  was  afforded  by  Manson  in  London.  About 
forty  mosquitoes  were  shipped  from  Rome  after  sucking  blood 
from  a  case  of  tertian  malaria,  and  Hanson's  son  allowed  himself 
to  be  bitten  by  them,  with  the  result  that  an  attack  of  tertian  fever 
followed  and  the  parasites  were  found  in  the  patient's  blood. 

Reproduction.  —  Both  man  and  mosquito  are  necessary  to 
complete  the  life  cycle  of  the  parasite.  Since  the  sexual  phase  is 
passed  within  the  mosquito  the  latter  is  considered  the  definitive 
host  and  the  former  the  intermediate  host.  It  is,  however,  more 
convenient  to  consider  the  asexual  cycle  first. 

Asexual  Phase  (Schizogony).  —  At  the  time  the  parasite  is 
conveyed  by  the  bite  of  the  mosquito  it  appears  as  a  small  ring- 
like  body,  and  on  entering  the  blood  stream  of  its  human  host 
it  attaches  itself  at  once  to  a  red  blood  corpuscle  and  commences 
to  send  out  distinct  pseudopodia  into  the  cell  substance.  As  it 
approaches  maturity  segmentation  begins,  giving  to  it  the  appear- 
ance of  a  rosette  within  the  corpuscle.  Very  soon  the  segments 
separate  into  small,  rounded  forms  or  merozoites,  the  cell  wall  of 
the  red  blood  corpuscle  ruptures,  and  the  young  merozoites  thus 
liberated  into  the  blood  stream  at  once  fasten  themselves  on  to 


292  BACTERIOLOGY   FOR  NURSES 

fresh  corpuscles  and  begin  anew  their  cycle  of  development.  The 
typical  malarial  chill  appears  just  at  the  time  of  the  freeing  of  the 
merozoites  (Fig.  45). 

tfot  all  of  the  full-grown  parasites  segment  and  produce  mero- 
zoites; a  certain  number  become  gametocytes  or  sexual  forms. 
The  female  cells,  the  macrogametocytes,  are  the  larger,  measuring 
about  12  fi,  and  containing  coarse  grains  of  pigment;  the  male 
cells,  microgametocytes,  are  smaller  and  stain  more  faintly.  In 


FIG.  45.  —  Malarial  Parasite.    A,  Development  of  Merozoites;  B,  Gametocytes. 

the  circulating  blood  of  human  beings  the  gametocytes  show  no 
further  change,  but  when  the  blood  is  shed  or  after  it  is  swallowed 
by  a  mosquito  an  interesting  series  of  changes  may  be  observed. 

Sexual  Phase  (Sporogeny).  —  The  same  phenomena  which 
occur  within  the  body  of  a  mosquito  may  be  observed  in  freshly 
drawn  infected  blood  examined  under  the  microscope.  In  the 
rounded  male  cell  a  vibratory  movement  of  the  chromatic  granules 
can  be  seen  and  very  soon  four  to  eight  long  flagella-like  appendages 
emerge.  These  delicate  filaments,  which  represent  the  sperm  cells 


PLASMODIA  293 

of  the  parasite,  are  extremely  active,  soon  become  detached,  and 
move  away  into  the  surrounding  fluid  in  search  of  the  female  cells. 
The  latter  project  a  small  portion  of  protoplasm  on  to  their  surface 
and  into  this  one  of  the  flagellate  sperm  cells  enters,  the  protoplasm 
is  instantly  retracted,  and  the  fertilized  female  cell  is  then  spoken 
of  as  a  zygote  or  ookinete.  In  the  stomach  of  a  mosquito  the 
zygotes  penetrate  the  stomach  wall  and  settle  between  the  muscle 
fibers,  where  on  the  second  day  after  the  ingestion  of  infected  blood 
they  may  be  seen  as  small,  rounded  cells  about  8  /*  in  diameter, 
containing  masses  of  pigment.  Around  each  zygote  a  membrane, 
termed  a  sporocyst,  develops,  and  as  the  organisms  increase  in 
size  they  project  into  the  stomach  cavity,  giving  to  it  an  irregular 
beaded  appearance.  Meanwhile  numerous  spherical  bodies  (sporo- 
blasts)  have  been  formed  within  the  interior  of  the  zygote  and 
these  again  divide  into  a  large  number  of  thread-like  cells  (sporo- 
zoites).  The  full  development  of  the  sporocyst  takes  from  eight 
to  ten  days,  after  which  it  bursts,  and  the  liberated  sporozoites  are 
carried  by  the  lymphatics  to  all  parts  of  the  body  of  the  mosquito. 
They  settle  especially  within  a  large  gland  which  is  a  combination 
of  a  poison  and  salivary  gland  and  which  also  is  in  close  connection 
with  the  biting  apparatus  of  the  mosquito.  Hence  when  an  infected 
mosquito  bites  a  human  subject  the  sporozoites  readily  pass  into 
the  puncture  with  the  gland  secretions  and  the  cycle  within  the 
human  host  begins.  Since  only  the  female  mosquito  sucks  blood 
it  alone  is  responsible  for  the  spread  of  the  disease. 

It  will  be  noted  that  in  the  mosquito  the  parasite  passes  through 
one  cycle  only,  while  in  the  human  host  it  passes  through  an  indefi- 
nite number  which  recur  at  regular  periods. 

So  far  as  is  known  the  parasites  of  human  malaria  do  not  invade 
any  other  mammalians  nor  does  any  other  species  of  mosquito 
other  than  Anopheles  harbor  them.  Anopheles  may  be  distin- 
guished from  the  more  common  Culex  by  the  following  character- 
istics :  it  appears  usually  after  sunset,  while  Culex  is  a  day-flying 
variety ;  when  at  rest  its  body  stands  off  at  an  acute  angle  from 
the  surface  of  the  object  on  which  it  is  reposing,  whereas  the  body 
of  Culex  appears  almost  parallel  with  the  surface.  Many  species 


294  BACTERIOLOGY  FOR  NURSES 

of  Anopheles  may  be  distinguished  by  their  spotted  wings.  Another 
distinction  exists  in  that  in  the  female  Anopheles  the  palpi  are  as 
long  as  the  proboscis,  whereas  in  the  female  Culex  they  are  always 
shorter.  The  eggs  and  larvae  of  the  two  genera  also  differ;  the 
eggs  of  Anopheles  are  single  and  are  supported  on  the  surface  of 
the  water  by  air  cells,  those  of  Culex  are  numerous  and  attached 
together  in  masses  by  a  cementing  substance.  The  breathing  tube 
of  the  Anopheline  larvae  is  short  and  its  angle  with  the  body 
necessitates  that  the  larvse  lie  parallel  with  the  surface  of  the 
water,  while  that  of  Culex  permits  the  body  to  lie  at  an  angle  with 
the  surface. 

Varieties  of  Malarial  Parasites.  —  Three  distinct  varieties  of 
the  malarial  parasite  have  been  described  :  Plasmodium  malaria  — 
the  cause  of  quartan  fever ;  Plasmodium  vivax —  the  cause  of  tertian 
fever ;  and  Plasmodium  falciparum  —  the  cause  of  estivo-autumnal 
fever.  Certain  authorities  are  of  the  opinion  that  two  varieties 
may  be  concerned  in  the  latter  condition.  Only  one,  however,  is 
definitely  known.  The  tertian  and  quartan  fevers  are  more  preva- 
lent in  temperate  countries  and  are  rarely  fatal ;  the  estivo-autum- 
nal type,  the  malignant  or  pernicious  fever  of  the  tropics,  is  of  a 
much  more  serious  nature  (Fig.  46). 

P.  Malarias.  —  The  asexual  cycle  of  development  is  completed 
in  seventy-two  hours,  the  typical  chill  and  fever  occurring  every 
third  day,  or  according  to  the  Roman  method  of  reckoning,  every 
fourth  day.  Double  or  even  triple  infection  may  occur.  In  the 
latter  case  infection  on  three  successive  days  would  result  in  the 
liberation  of  a  brood  of  merozoites  and  the  consequent  clinical 
symptoms  every  day.  The  young  P.  malariae  are  less  active  than 
P.  vivax ;  the  pigment  is  of  a  darker  color,  more  coarsely  granular, 
and  is  arranged  around  the  periphery  of  the  parasite,  while  that 
in  the  tertian  parasite  is  distributed  throughout  the  protoplasm. 
P.  malarise  arranges  itself  as  a  band  across  the  infected  corpuscle. 
When  mature  the  adult  forms  do  not  exceed  the  size  of  the  red 
blood  corpuscles,  and  the  segments,  usually  six  to  twelve  in  number, 
are  arranged  symmetrically  around  the  central  pigment,  giving  the 
parasite  at  this  stage  the  so-called  daisy  appearance. 


PLASMODIA 


295 


P.  Vivax.  —  The  parasite  of  tertian  malaria  completes  its  asexual 
development  in  forty-eight  hours;  the  paroxysms  accordingly 
occur  on  alternate  days.  The  adult  parasite  is  larger  and  shows 


CULEX. 


ANOPHELES 


FIG.  46.  —  Comparison  between  Culex  and  Anopheles. 

more  active  ameboid  movements  than  the  quartan  form.  The 
average  number  of  merozoites  produced  is  usually  about  sixteen. 
P.  Falciparum.  —  The  developmental  cycle  in  the  human  host 
occupies  from  twenty-four  to  forty-eight  hours.  The  parasite  is 
much  smaller  than  the  two  other  varieties  and  rarely  reaches  more 


296  BACTERIOLOGY  FOR  NURSES 

than  half  the  diameter  of  a  red  blood  corpuscle.  The  gametocytes 
differ  also  in  that  they  are  crescentic  in  shape  when  first  liberated 
from  the  red  blood  cells. 

Methods  of  Examination.  —  The  organisms  may  be  studied 
in  fluid  or  dried  preparations.  In  the  former  case  a  drop  of  blood 
from  a  pricked  finger  or  ear  lobe  is  allowed  to  fall  upon  a  coverslip 
and  examined  in  a  hanging  drop,  or  the  coverslip  may  be  gently 
inverted  over  a  flat  slide  and  the  rims  sealed  with  vaseline.  Dried 
preparations  are  made  in  the  manner  described  for  blood  films 
and  stained  with  one  of  the  special  blood  stains. 

Cultivation.  —  In  1911  Bass  and  Johns  announced  that  they 
had  succeeded  in  cultivating  P.  vivax  and  P.  falciparum  outside 
of  the  body.  The  first  cultures  were  obtained  by  defibrinating 
blood  taken  from  malarial  patients.  Growth  of  the  parasites  took 
place  within  the  red  blood  corpuscles  in  exactly  the  same  manner 
as  in  the  human  body  so  long  as  anaerobic  conditions  were  main- 
tained. Parasites  transferred  from  tube  to  tube  of  defibrinated 
blood  were  thus  carried  through  several  generations. 

Pathogenesis.  —  Relatively  little  is  known  of  the  pathogenic 
processes  which  occur  in  malaria.  The  organisms  are  not  always 
equally  abundant  in  the  circulating  blood ;  at  certain  stages  they 
tend  to  be  more  numerous  in  the  internal  organs.  The  spleen  be- 
comes enlarged.  The  liver  and  kidneys  may  also  show  some 
swelling.  It  is  definitely  known  that  the  occurrence  of  the  fever 
is  coincident  with  the  liberation  of  the  merozoites,  but  whether  the 
increased  temperature  is  due  to  a  toxin  liberated  at  the  same  time 
is  as  yet  undetermined.  Since  the  organisms  attack  mainly  the 
red  blood  corpuscles  the  anemia  which  is  so  pronounced  a  feature 
of  the  disease  is  readily  explained :  the  parasite  absorbs  the  cell 
pigment  and  thus  destroys  its  function.  During  the  paroxysms 
there  is  always  a  marked  increase  in  the  number  of  leukocytes, 
followed  by  a  rapid  decline.  An  interesting  feature  of  the  disease 
is  the  increased  percentage  of  the  mononuclear  leukocytes,  which 
sometimes  even  outnumber  the  polynuclears.  The  cells  become 
particularly  active  phagocytes  and  engulf  great  numbers  of  pig- 
mented  parasites.  In  fact,  the  presence  of  excessive  numbers  of 


PLASMODIA  297 

pigmented  mononuclear  leukocytes  has  been  taken  as  evidence  of 
malaria  by  some  workers,  even  though  no  parasites  could  be  found 
in  the  blood.  A  striking  feature  in  estivo-autumnal  fever  is  the 
presence  of  enormous  numbers  of  infected  red  blood  cells  in  the 
capillaries  of  the  brain  and  abdominal  viscera. 

Immunity.  —  Many  mild  cases  recover  without  treatment 
hence  it  is  evident  that  some  immunity  is  produced  by  an  infection. 
It  is  temporary,  however,  and  does  not  protect  against  reinfection. 

Prophylaxis.  —  Since  malarial  infection  is  transferred  only  by 
a  certain  species  of  mosquito  a  malarial  patient  can  be  considered 
a  source  of  danger  only  when  that  particular  variety  of  mosquito 
is  in  the  vicinity.  Conversely,  the  Anopheles  mosquitoes  are  harm- 
less if  they  have  never  had  the  opportunity  of  drinking  blood 
containing  gametocytes  from  a  malarial  patient.  With  the 
knowledge  of  the  manner  in  which  infection  is  spread  active 
measures  have  been  taken  in  different  countries  to  control  the 
disease,  the  most  efficacious  of  which  is  the  extirpation  of  the  mos- 
quitoes by  the  suppression  of  their  breeding  places.  Since  Anoph- 
eles breeds  in  open  ponds  and  natural  collections  of  water  in 
fields  and  swamps  this  may  be  accomplished  by  filling  up  low  places 
and  drying  the  surface  of  land  with  drains.  Where  drainage  is 
not  practical  the  number  of  mosquitoes  may  be  kept  in  check  by 
introducing  fish  into  ponds  and  other  collections  of  water.  Upon 
limited  surfaces  the  larva  may  be  destroyed  by  cutting  off  the  air 
supply  by  means  of  a  thin  layer  of  coal  oil. 

Koch  advocated  the  administration  of  quinine  in  order  to  destroy 
the  parasite  within  the  human  body  and  thus  prevent  the  infec- 
tion of  the  mosquito.  The  drug  has  a  remarkable  effect  upon 
the  merozoites  of  the  tertian  and  quartan  types  and  great  success 
has  resulted  from  its  use.  A  single  large  dose  may  be  administered 
as  the  temperature  begins  to  decline,  that  is,  shortly  after  the 
young  merozoites  have  been  liberated  into  the  blood  stream ;  or  in 
cases  of  double  or  triple  infection  where  different  broods  come  to 
maturity  at  different  times  smaller  doses  at  definite  intervals 
have  given  better  results.  Unfortunately,  the  gametocytes  are 
quite  resistant  to  the  drug. 


298  BACTERIOLOGY  FOR  NURSES 

The  use  of  quinine  as  a  prophylactic  on  a  large  scale  is  a  com- 
paratively recent  measure.  The  Italian  Government  in  1902 
commenced  its  sale  at  cost  price  to  those  communities  which  agreed 
to  distribute  it  gratuitously  to  individuals  unable  to  purchase  it. 
The  result  has  been  remarkable.  During  the  ten  years  previous 
to  1902  the  deaths  from  malaria  averaged  14,048  annually,  whereas 
during  the  ten  years  following  the  average  fell  to  3853. 

The  administration  of  quinine  to  healthy  individuals  does 
not  prevent  infection  ;  it  destroys  the  young  parasites  in  the  blood 
after  infection  has  occurred.  Its  use  is  advantageous  in  that  it 
is  cheap  and  its  action  is  prompt.  It  can,  however,  only  be  consid- 
ered as  a  tentative  measure  and  cannot  supplant  mosquito  sup- 
pression. 

BLACKWATER  FEVER 

The  condition  known  as  blackwater  fever  occurs  especially 
amongst  Europeans  in  tropical  countries.  It  is  characterized 
by  fever,  hemoglobinuria,  and  delirium,  frequently  ending  in  coma 
and  death.  The  etiology  of  the  disease  is  not  at  all  clear.  A 
few  observers  consider  it  an  independent  disease;  the  majority, 
however,  believe  it  to  be  the  terminal  stage  of  a  malarial  infection. 
The  fact  that  an  attack  is  often  precipitated  by  the  administra- 
tion of  quinine  has  led  to  the  suggestion  that  the  drug  may  be  the 
responsible  agent  for  the  marked  destruction  of  red  blood  cells 
which  characterizes  the  disease.  In  the  great  majority  of  cases, 
however,  if  the  malarial  organisms  cannot  be  found  in  the  blood 
there  is  evidence  of  the  patient  having  suffered  from  repeated 
attacks  of  malaria. 

CILIATA 

The  Ciliata  are  the  highest  type  of  protozoa.  They  may  be 
distinguished  from  the  other  groups  by  the  presence  of  cilia  dis- 
tributed over  the  cell,  which  serve  as  organs  of  locomotion  and 
which  give  to  the  group  its  name.  They  possess  special  structures 
for  the  reception  of  food  and  also  for  excreting  waste  products. 

Only  one  of  the  ciliates,  Balantidium  coli,  has  been  found  patho- 


CILIATA  299 

genie  for  man.  The  parasite  was  first  described  in  1857;  it  is 
frequently  present  in  the  intestinal  tract  of  swine,  and  though 
usually  harmless  it  may  give  rise  in  them  to  a  subacute  form  of 
dysentery.  Cases  of  human  infection  have  been  reported,  most  of 
which  have  been  found  to  be  suffering  from  chronic  intestinal 
catarrh. 

The  organism  has  somewhat  the  form  of  an  egg  with  a  funnel- 
shaped  mouth  opening.  The  ectoplasm  is  covered  with  thick 
bands  of  cilia  which  give  the  organism  a  striated  appearance. 
Multiplication  usually  takes  place  by  binary  division ;  conjuga- 
tion also  occurs. 

CHLAMYDOZOA 

A  small  group  of  minute  coccus-like  organisms  have  been  de- 
scribed which  have  the  power  of  enveloping  themselves  with  a 
covering  derived  from  the  cell  substance  of  the  host.  Certain 
authorities  have  created  a  special  class  named  chlamydozoa  for 
them  (Greek  stem,  chlamys  —  a  mantle)  and  include  them  with 
the  protozoa.  A  number  of  observers  believe  that  members  of 
the  group  may  be  the  causal  agents  of  rabies,  smallpox,  scarlet 
fever,  trachoma,  and  other  diseases.  No  definite  proof,  however, 
has  as  yet  been  advanced. 


CHAPTER  XXVIII 

DISEASES   CAUSED    BY    FILTRABLE    VIRUSES.      DIS- 
EASES  OF   UNKNOWN   ETIOLOGY 

A  NUMBER  of  infectious  diseases  are  caused  by  organisms  that 
are  able  to  pass  through  a  fine  porcelain  filter.  That  the  causal 
agents  are  present  in  the  filtrate  has  been  proven  by  animal  in- 
oculations ;  yet  in  a  certain  number  of  cases  the  highest  degree  of 
magnification  possible  fails  to  reveal  their  presence.  Such  organ- 
isms evidently  are  either  "  ultramicroscopic  "  or  they  cannot 
be  rendered  visible  by  present  methods.  Just  what  determines 
the  ability  of  an  organism  to  pass  through  a  filter  is  uncertain ; 
plasticity  may  be  as  important  a  factor  as  minuteness.  A  few 
of  the  filtrable  viruses,  although  exceedingly  minute,  are  still 
within  the  range  of  visibility. 

The  groups  apparently  bear  no  close  relationship  one  to  the 
other.  Their  modes  of  transmission  are  also  widely  different.  Cer- 
tain forms,  for  example,  are  transmitted  by  biting  insects,  as  in 
yellow  fever ;  others  by  direct  contact  through  a  wound  or  abrasion, 
as  in  rabies ;  still  others  by  contact,  as  in  cattle  plague.  Eventually, 
certain  members  will  in  all  probability  be  classified  with  the  pro- 
tozoa and  others  with  the  bacteria. 

In  order  to  be  certain  that  an  organism  is  filtrable  several  pre- 
cautions must  be  observed  in  carrying  out  the  filtration  process. 
The  integrity  of  the  filter  must  first  be  tested  with  a  culture  of 
known  infiltrable  organisms,  all  of  which  must  be  retained  and 
not  pass  into  the  filtrate ;  after  which  the  filter  must  be  sterilized 
to  insure  its  freedom  from  all  germs.  The  pressure  or  suction 
should  only  be  moderate ;  and  the  filtration  should  be  completed 
within  two  hours ;  otherwise  certain  bacteria  might  grow  through 

300 


EPIDEMIC   POLIOMYELITIS  301 

rather  than  pass  through  the  pores.  After  all  precautions  have 
been  taken  itimust  be  shown  that  the  pathogenicity  of  the  filtrate 
is  due  to  a  living  organism  and  not  a  toxin.  This  may  be  deter- 
mined by  inoculating  a  series  of  animals  with  the  filtrate  and  later 
inoculating  another  series  of  animals  with  a  filtrate  of  material 
obtained  from  the  first  series. 

Epidemic  Poliomyelitis.  —  The  disease  occurs  both  sporadically 
and  in  epidemic  form  in  all  quarters  of  the  world  and  affects  chiefly 
children  under  five  years  of  age.  The  mortality  is  low,  but  about 
75  per  cent  of  the  survivors  are  permanently  deformed.  The 
chief  symptoms  of  the  disease  are  fever,  sometimes  accompanied 
by  sore  throat  and  followed  after  a  few  days  by  paresis  and 
paralysis. 

Nothing  was  definitely  known  of  the  etiology  of  the  disease 
until,  in  1909,  Landsteiner  and  Popper  in  Vienna  reported  its 
transmission  to  apes  by  the  intraperitoneal  injection  of  an  emulsion 
of  the  spinal  cord  of  a  child  who  had  died  of  infantile  paralysis  on 
the  fourth  day  of  illness.  In  the  same  year  Flexner  and  Lewis 
transmitted  the  disease  to  monkeys  by  intracerebral  injections 
and  found  that  the  brain  and  cord  of  the  inoculated  animals  were 
infective  for  other  monkeys. 

In  1911  Flexner  and  Noguchi  announced  that  they  had  succeeded 
in  cultivating  the  virus  in  human  ascitic  fluid  to  which  had  been 
added  a  small  piece  of  fresh  sterile  rabbit  kidney.  Growth  takes 
place  at  first  only  under  anaerobic  conditions.  Fragments  of  in- 
fected brain  or  cord  or  a  filtrate  of  nerve  tissue  may  be  used  for 
inoculating  the  medium.  After  about  five  days'  incubation  at 
37°  C.  growth  appears  as  an  opalescent  haze  upon  the  fragment 
of  tissue.  Film  preparations  treated  with  Giemsa  stain  show  the 
organisms  as  minute  bluish  or  violet  round  bodies,  measuring  about 
0.2  p  in  diameter  and  arranged  in  pairs,  chains,  or  groups.  They 
appear  to  have  a  special  affinity  for  nerve  tissue.  In  an  infection 
they  are  contained  in  the  brain,  spinal  cord,  salivary  glands,  mucous 
membranes  of  the  nasopharynx,  and  very  rarely  in  the  cerebro- 
spinal  fluid  or  the  blood.  They  are  not  weakened  by  freezing 
and  will  withstand  1  per  cent  carbolic  acid  for  at  least  five  days. 


302  BACTERIOLOGY  FOR  NURSES 

They  are,  however,  only  moderately  resistant  to  heat ;  exposure 
to  45°  C.  to  50°  C.  for  half  an  hour  destroys  them. 

Experimentally  the  disease  may  be  induced  in  monkeys  by 
intracranial  inoculation  or  by  rubbing  the  virus  on  the  sound  nasal 
membrane.  In  such  cases  the  incubation  period  averages  from 
eight  to  nine  days,  but  may  range  from  five  to  forty  days.  Mon- 
keys which  have  recovered  from  an  attack  of  the  disease  are  im- 
mune to  fresh  inoculations ;  also  it  has  been  shown  that  the  serum 
of  such  animals  when  mixed  with  infective  material  has  the  power  of 
neutralizing  the  virus  to  a  certain  extent.  Furthermore,  the  serum 
of  recently  recovered  human  cases  when  injected  into  new  cases 
within  the  first  forty-eight  hours  is  capable  of  arresting  paralysis. 

Since  the  worst  epidemics  occur  in  summer  and  in  rural  rather 
than  urban  communities  the  view  has  been  advanced  that  an 
insect  may  be  responsible  for  its  transmission.  Experiments  have 
shown  that  a  blood-sucking  fly  when  infected  may  transmit  the 
virus  to  monkeys.  It  is  doubtful,  however,  if  the  fly  is  responsible 
for  the  spread  of  the  disease ;  there  is  more  evidence  at  the  present 
time  that  its  dissemination  is  due  to  contact,  direct  or  indirect, 
with  the  virus  discharged  in  the  secretions  from  mouth  or  nose. 
The  existence  of  healthy  carriers  may  perhaps  explain  the  outbreak 
of  the  various  epidemics. 

Yellow  Fever.  —  The  disease  is  an  acute  infection  occurring 
chiefly  in  tropical  countries  and  characterized  by  fever,  jaundice, 
and  hemorrhages.  Several  investigations  have  been  made  con- 
cerning its  etiology,  the  most  extensive  probably  being  that  of  the 
United  States  Commission.  The  members  of  the  Commission, 
Reed,  Carroll,  Agramonte,  and  Lazear,  began  their  work  in  1901, 
and  although  they  did  not  succeed  in  demonstrating  the  causal 
agent  they  discovered  facts  concerning  the  transmission  of  the 
disease  that  has  led  practically  to  its  eradication  in  areas  where 
the  necessary  precautions  have  been  taken. 

In  Havana  preventive  measures  were  first  enforced  in  1901, 
and  within  ninety  days  the  town  was  free  of  yellow  fever.  Several 
weeks  later  new  cases  appeared,  but  the  same  measures  were  ap- 
plied and  the  disease  was  quickly  stamped  out. 


RABIES  303 

The  Commission  demonstrated  that  two  species  of  hosts  are 
necessary  for  the  life  cycle  of  the  parasite,  human  beings  and 
mosquitoes,  and  that  under  natural  conditions  it  is  transmitted 
from  infected  to  healthy  individuals  only  by  the  bite  of  a  small 
mosquito,  JEdes  calopus,  first  spoken  of  as  Stegomyia.  After  being 
bitten  a  period  of  about  five  days  elapses  before  the  parasite  appears 
in  the  blood  of  the  individual  and  remains  there  only  during  the 
three  succeeding  days.  Thus  an  infected  mosquito  must  necessarily 
have  sucked  the  blood  of  a  patient  during  the  first  three  days  of 
his  illness.  An  infected  mosquito  does  not  transmit  the  parasite 
until  at  least  twelve  days  after  it  has  bitten  the  first  patient. 
Yellow  fever  can  be  produced  in  man  under  artificial  conditions 
by  injecting  the  blood  of  a  patient  taken  during  the  first  three  days 
of  illness  or  even  by  inoculation  with  the  serum  of  such  a  patient 
after  it  has  passed  through  a  fine  Berkefeld  filter. 

Noguchi  has  recently  announced  the  visibility  of  the  parasite 
by  dark  field  illumination  and  the  possibility  of  its  culture. 

Rabies  or  Hydrophobia.  —  The  disease  occurs  occasionally 
amongst  most  carnivora.  It  is  transmitted  to  man  usually  by  the 
bite  of  a  dog.  Experiments  have  shown  that  the  virus  is  contained 
in  the  saliva  from  twenty-four  to  forty-eight  hours  before  symp- 
toms appear.  In  man  the  incubation  period  after  being  bitten 
varies  from  fourteen  days  to  seven  months,  the  rapidity  with  which 
the  disease  develops  being  governed  by  the  amount  of  virus 
introduced,  the  point  of  inoculation,  and  the  individual  degree  of 
susceptibility.  It  has  been  frequently  observed  that  in  wounds 
inflicted  where  the  skin  is  thick  and  the  nerves  are  few,  or  where 
the  clothing  has  afforded  some  degree  of  protection,  the  incubation 
period  is  relatively  long,  whereas  in  wounds  in  parts  more  abun- 
dantly supplied  with  nerves  the  incubation  period  is  much  shorter 
and  the  disease  usually  more  virulent.  The  symptoms  of  rabies 
generally  begin  with  pain  in  the  wound,  extending  along  the  nerves 
in  the  limb  bitten,  followed  by  a  stage  of  nervous  irritability,  diffi- 
culty in  breathing  and  swallowing  due  to  spasmodic  contraction 
of  the  throat  muscles,  and  a  marked  increase  in  the  flow  of  saliva. 
Very  soon  the  convulsive  attacks  become  more  or  less  general 


304  BACTERIOLOGY  FOR  NURSES 

over  the  whole  body.  Generally  the  patient  has  full  consciousness 
between  the  attacks  until  the  final  stage  of  the  disease.  The 
convulsive  period  lasts  from  one  to  four  days  and  may  be  followed 
by  a  paralytic  stage  lasting  from  two  to  eighteen  hours.  In  the 
majority  of  cases  death  occurs  on  the  third  or  fourth  day  after 
symptoms  appear. 

Despite  repeated  investigations  all  attempts  to  discover  the 
causal  agent  of  the  disease  were  unsuccessful  until  in  1903  Negri 
described  certain  round  or  angular  bodies  lying  within  the  large 
nerve  cells  or  their  processes,  which  he  claimed  were  specific  for 
rabies  and  in  all  probability  protozoan  in  character.  Negri's 
observations  have  been  generally  confirmed  and  the  presence  of 
the  bodies  is  accepted  as  diagnostic. 

Negri  bodies  may  be  detected  in  fresh  tissue  by  means  of  the 
smear  method.  Small  pieces  of  gray  matter  are  removed  from 
three  different  portions  of  the  central  nervous  system :  (1)  from 
the  cortex  in  the  region  of  the  crucial  sulcus ;  (2)  from  Ammon's 
horn,  and  (3)  from  the  gray  matter  of  the  cerebellum.  Minute 
portions  of  the  tissue  are  placed  on  well-cleaned  slides,  crushed 
into  a  thin  layer  under  a  coverslip,  after  which  the  coverslip  is 
moved  slowly  and  evenly  along  the  slide,  leaving  a  film  of  nerve 
cells  in  its  train.  The  smears  are  dried  in  the  air  and  stained  with 
Giemsa's  stain.  When  examined  under  the  oil  immersion  lens 
the  organisms  appear  pale  blue  and  within  their  protoplasm  one 
or  several  round  or  oval  pink  bodies  may  be  seen.  In  addition, 
both  within  the  protoplasm  and  the  pink-stained  bodies  small  red 
or  violet  granules  occur  singly  or  in  clumps. 

Experiments  have  shown  that  the  virus  is  filtrable.  It  is  un- 
harmed by  freezing.  On  the  other  hand,  it  is  readily  destroyed 
by  drying  and  by  direct  sunlight  and  is  rendered  inert  by  exposure 
for  one  hour  to  50°  C.  When  protected  from  heat,  sunlight,  and 
air  it  retains  its  virulence  for  a  long  period. 

Pasteur  in  1880  made  the  important  discovery  that  rabies 
may  be  prevented  by  immunization  with  gradually  increasing 
doses  of  the  attenuated  virus.  So  successful  was  his  method  of 
treatment  that  with  some  modifications  it  is  still  used  in  all  parts 


RABIES  305 

of  the  world.  The  method  is  based  upon  the  principle  of  stimulat- 
ing the  production  of  rabic  antibodies  by  the  injection  of  weakened 
virus  during  the  period  of  incubation,  so  that  the  virus  introduced 
into  the  wound  may  be  destroyed.  Starting  with  the  idea  that 
the  virus  as  it  occurs  in  rabid  dogs  under  natural  conditions  (street 
virus)  is  of  a  more  or  less  constant  degree  of  virulence,  he  found 
that  the  potency  of  such  a  virus  could  be  diminished  within  a  cer- 
tain limit  by  passage  through  monkeys  and  similarly  increased 
by  passage  through  rabbits.  In  the  latter  case  the  virulence 
could  be  so  exalted  for  rabbits  that  the  incubation  period  was 
lessened  from  twelve  or  fourteen  days  to  six  or  seven  days,  but 
beyond  that  point  it  was  impossible  to  go.  A  virus  of  such  strength 
he  termed  "  fixed."  By  drying  fixed  virus  over  caustic  potash 
he  was  able  to  obtain  different  degrees  of  attenuation  according 
to  the  length  of  time  of  exposure,  the  weakest  virus  being  so  modi- 
fied that  it  could  not  produce  the  disease  in  man  but  yet  was  able 
to  produce  the  specific  antibodies. 

"  Fixed  "  virus  is  prepared  by  the  subdural  inoculation  of  street 
virus  through  a  series  of  young  rabbits.  During  from  thirty  to 
fifty  passages  the  incubation  period  is  reduced  to  six  or  seven 
days.  Immediately  on  the  death  of  the  animal  a  piece  of  the 
floor  of  the  fourth  ventricle  is  emulsified  in  sterile  broth  and 
three  or  four  drops  of  the  emulsion  are  injected  beneath  the  dura 
of  a  normal  rabbit.  In  from  six  to  seven  days  paralytic  symptoms 
appear  followed  in  from  three  to  four  days  by  death.  The  cord 
and  brain  are  then  removed  under  aseptic  precautions  and  the 
brain  is  set  aside  for  further  animal  inoculations.  From  the  cord, 
which  is  severed  just  below  the  medulla,  a  fragment  is  cut  off  and 
dropped  into  sterile  broth  to  test  for  purity  and  the  remainder 
is  then  divided  into  two  equal  portions  which  are  suspended  by 
sterilized  silk  threads  in  a  glass  jar  containing  sticks  of  caustic 
potash.  The  jars  with  the  suspended  cords  are  kept  in  a  dark 
room  at  a  temperature  of  about  22°  C.  After  a  suitable  period  of 
drying  small  pieces  of  cord  are  emulsified  and  injected  subcutane- 
ously  into  patients  under  treatment.  In  the  New  York  Board 
of  Health  Laboratories  £  c.c.  of  the  indicated  cord  is  emulsified 


300  BACTERIOLOGY   FOR  NURSES 

in  3  c.c.  of  saline,  and  2J  c.c.  of  thin  emulsion  in  administered.    If 

the  material  is  In  IM-  flipped,  'JO  per  cent  glycerin  mid  <)..r>  percent 

carbolic  acid  is  added. 
The  uniform  dose  of  2£  c.c.  for  adults,  prepared  as  described 

al>o\r,  i  .  ;i<lmi!n  ,trml  in  a  scries  of  inoculations  as  follows: 

I  K>  Ann  <<r   <  '"in. 

lii-Ml     ............  H  and  7  and  0  dayti 

Hrcond       ......     .     ,     •     .     .  4  and  H  dnyH 

'I  I  in  il    .......    ii(.    .  fi  and  4  dnyn 

I"  'irlli  .........     ,     ,     .  ,'i  <lny« 

I  nil,     ........     ,,,.3  day* 

Sixth     ........     ,     ,     ,     .  2  day* 

S.-vmil.     .......     ,    ...    ,     .  2  days 

I  ,ir  I,  ill  ........     ,     .     .     .  I  day 

\inil,        .......    ,    ,    ...  fidayif 

Tenth  ......    •..,,,.  4  days 

I'Jrvriitli  1   iliiVH 

Tw.-mi,  .......    3  days 

n.iruwnth     ..........    3d»y» 

hilirtrrlilli  '.! 


Sixlrrnlli  J   <|MVH 

Srvi-nlrrnlli  ......      ItdiiyM 

I'JUlllci'lllli  ......        '.!   illlVH 

Niiii-tiM-iil.il     ..........     8  <layn 

Turntirlli  :•  ,|MVM 

Twi'iity-flrnt  ......    i    i    •    .     1  day 

According  to  relinhle  stutisties  the  inorlnlity  of  riil)ies  witlnml 
tin-  I'nsleiir  trenhiieiif  is  iihoiit  ll'»  per  cent,  with  the  treiittneiit  0.4(1 
per  cent.  'Puking  i'llo  coiisidcrjil  ion  only  those  enses  in  which 
the  diagnosis  of  rabies  has  been  confirmed  in  the  imimal,  the 
mortidity  of  the  eases  treated  at  the  Pasteur  Institute  in  I'aris 
for  the  pnst  ten  years,  which  number  about  (M)00,  has  been  only 
(Ml  per  cent,  an  enormous  reduction  in  the  Hi  per  cent  of  untreated 
eases. 

Once  the  symptoms  of  rabies  have  appeared  the  treatment  is 
iiiinviiiliii^. 

An  antirabie  serum  has  been  produced  by  inoculating  sheep 
or  horses  with  "  fixed  "  virus.  Its  use  alone  is  of  little  effect. 
Favorable  results  have  followed  its  administration  in  conjunction 
with  the  Pasteur  treatment,  its  use  enabling  the  injection  of  (lie 
virus  to  be  condensed. 


TRENCH   FEVER  307 

Foot  and  Mouth  Disease.  —  The  disease,  which  was  probably 
the  first  shown  to  be  due  to  a  filtrable  virus,  is  highly  infectious 
and  occurs  chiefly  among  cattle.  It  may,  however,  be  communi- 
cated to  man  by  milk  or  milk  products  or  by  contact  with  infected 
animals.  It  is  characterized  by  a  vesicular  eruption  on  the  mucous 
membrane  of  the  mouth  and  on  the  skin  of  the  foot.  No  specific 
microorganism  has  been  demonstrated.  It  has  been  found,  how- 
ever, that  lymph  from  the  vesicles  passed  through  the  finest  porce- 
lain filter  is  still  infectious. 

Dengue.  —  The  disease  is  restricted  to  warm  climates  and  in 
many  respects  resembles  yellow  fever.  The  virus  is  filtrable, 
is  found  in  the  blood  of  patients  suffering  from  the  disease  on  the 
third  and  fourth  day,  and  is  probably  transmitted  by  a  mosquito. 

Trench  Fever.  —  During  the  recent  war  the  malady  has  become 
recognized  as  a  distinct  disease.  It  is  characterized  by  a  sudden 
onset,  pains  in  the  limbs,  back,  and  behind  the  eyes,  fever,  head- 
ache and  giddiness.  After  from  three  to  six  days  the  symptoms  sub- 
side. Frequently,  however,  in  three  or  four  days  there  is  a  relapse 
of  shorter  duration  and  milder  form  than  that  of  the  original  attack. 
In  the  majority  of  cases  complete  recovery  occurs ;  occasionally 
chronic  rheumatic  pains  and  myalgia  persist. 

Extensive  studies  have  shown  that  the  parasite  is  present  in 
the  blood,  urine,  and  sputum  of  infected  individuals,  and  that  it 
is  at  least  in  one  stage  able  to  pass  through  a  moderately  fine  filter. 
It  is  somewhat  resistant  to  drying  and  to  sunlight,  but  is  killed 
by  exposure  to  a  temperature  of  70°  C.  for  half  an  hour.  It  is 
transmitted  by  lice. 

A  number  of  other  diseases  affecting  animals  have  been  shown 
to  be  due  to  filtrable  agents,  among  which  are  cattle  plague  (Rin- 
derpest), hog  cholera,  African  horse  sickness,  chicken  sarcoma,  and 
contagious  pleuropneumonia  of  cattle.  A  plant  disease,  the 
Mosaic  disease  of  tobacco,  has  also  been  shown  to  be  due  to  a 
filtrable  virus. 

Smallpox.  —  Variola  or  smallpox  has  been  one  of  the  most 
studied  of  the  infectious  diseases,  not  only  on  account  of  the  terrible 
havoc  it  formerly  wrought,  but  also  to  furnish  an  explanation  of 


308  BACTERIOLOGY   FOR  NURSES 

the  active  immunization  resulting  from  the  form  of  vaccination 
introduced  by  Jenner. 

The  origin  of  vaccination  against  smallpox  is  not  definitely 
known.  The  method  of  introducing  the  virus  from  a  smallpox 
patient  into  a  healthy  person  through  an  abrasion  of  the  skin 
in  order  to  produce  a  mild  form  of  the  disease  and  protection 
against  subsequent  attacks  was  practiced  by  the  Turks  during  the 
eighteenth  century.  In  1778  Lady  Mary  Montagu,  the  wife  of 
the  British  Ambassador  at  Constantinople,  observing  this  practice 
among  the  Turks,  had  her  own  son  and  daughter  inoculated  and 
by  her  influence  was  instrumental  in  introducing  the  practice  in 
Europe.  The  disease  thus  induced  was  generally  but  not  always 
mild.  Occasionally  a  case  of  unexpected  virulence  developed  which 
proved  fatal  to  the  individual  inoculated  and  a  starting  point  of 
infection  among  unprotected  persons. 

Edward  Jenner,  a  physician  practising  in  Gloucestershire, 
England,  was  much  impressed  with  the  popular  belief  that  those 
who  contracted  cowpox  from  an  affected  animal  were  immune 
to  subsequent  infection  from  smallpox.  He  believed  that  a  disease 
occurring  amongst  horses,  known  as  horsepox,  manifested  by  an 
inflammatory  and  ulcerative  condition  of  the  hocks,  was  transferred, 
by  the  hands  of  men  who  dressed  the  sores,  to  the  teats  of  the  cows 
later  milked  by  them  and  gave  rise  to  cowpox.  From  infected 
cows  other  milkers  contracted  a  mild  form  of  the  disease  which 
manifested  itself  in  lesions  similar  to  those  in  the  cow ;  namely, 
slight  fever,  malaise,  loss  of  appetite,  and  a  local  papular  eruption, 
later  becoming  pustular  and  finally  drying  up,  leaving  cicatrices 
varying  in  depth  and  extent  at  their  site.  Fully  convinced  that 
such  an  infection  gave  rise  to  immunity  against  smallpox  Jenner 
determined  to  make  experimental  tests.  In  May,  1796,  heinoculated 
a  boy  with  lymph  from  a  cowpox  lesion  on  the  hand  of  a  dairymaid 
and  in  July  he  inoculated  the  same  boy  with  pus  from  a  smallpox 
patient  without  resulting  infection.  In  1798  he  furnished  addi- 
tional proof  of  the  protection  afforded  by  vaccination  with  cowpox 
virus  by  inoculating  a  child  direct  from  the  vesicle  on  the  teat  of 
a  cow  and  from  the  resulting  lesion  inoculating  another  child,  and 


SMALLPOX  309 

so  on  through  a  series  of  five  children,  after  which  all  were  inoculated 
with  smallpox  virus  without  a  single  case  developing.  So  con- 
vincing were  Jenner's  experiments  that  within  a  year  or  two  such 
vaccination  became  extensively  practiced  all  over  Europe.  It  is 
said  to  have  been  introduced  into  the  United  States  in  July,  1800, 
by  Dr.  Benjamin  Waterhouse,  Professor  of  Physic  at  Harvard 
University,  who  vaccinated  his  own  children. 

Jenner  and  his  supporters  met  with  bitter  opposition,  and  even 
now,  more  than  one  hundred  years  later,  there  are  still  to  be  found 
opponents  to  cowpox  vaccination,  notwithstanding  the  fact 
that  its  systematic  application  would  soon  eradicate  smallpox  from 
the  list  of  human  diseases. 

The  relationship  of  smallpox  (variola)  to  cowpox  (vaccinia) 
has  been  the  subject  of  a  great  deal  of  controversy  since  Jenner's 
time,  yet  no  adequate  explanation  has  been  found.  According 
to  the  general  belief  the  smallpox  virus,  whatever  it  may  be,  is 
so  modified  by  its  passage  through  a  lower  animal  that  it  loses 
forever  its  power  of  producing  smallpox,  yet  it  still  retains  the 
ability  to  provoke  the  production  of  antibodies  protective  against 
the  disease. 

In  sections  of  skin  from  both  variola  and  vaccinia  microscopic 
cell  inclusions  were  first  described  by  Guarnieri  in  1892.  These 
"  vaccine  bodies  "  are  thought  by  certain  investigators  to  be 
protozoan  in  character  and  to  be  closely  associated  with  the 
cause  of  both  variola  and  vaccinia ;  other  authorities  regard  them 
as  degenerative  products. 

As  yet  all  attempts  to  obtain  growth  of  the  virus  on  artificial 
culture  media  has  been  unsuccessful.  A  few  investigators  have 
reported  that  the  virus  is  filtrable ;  others  have  failed  after  re- 
peated efforts  to  obtain  an  infective  filtrate. 

During  the  early  days  of  Jennerian  vaccination  it  was  custom- 
ary to  inoculate  with  the  material  taken  from  the  pustules  of 
those  previously  vaccinated  with  cowpox  lymph.  The  procedure 
served  the  purpose  of  immunization  but  it  had  several  drawbacks, 
the  chief  of  which  was  the  danger  of  transmitting  syphilis.  For 
many  years  now  it  has  been  the  custom  to  employ  only  vaccine 


310  BACTERIOLOGY  FOR  NURSES 

obtained  directly  from  healthy  animals,  the  production  of  which 
can  be  carefully  controlled  and  tested. 

The  virus  used  for  vaccinating  the  animals  (seed  virus)  may 
be  prepared  in  several  ways.  That  usually  employed  by  the  New 
York  City  Health  Department  is  obtained  by  first  passing  an 
emulsion  of  crusts  obtained  from  healthy  children  about  nineteen 
days  after  vaccination  through  a  calf  and  subsequently  through 
rabbits.  The  pulp  obtained  from  the  rabbit  lesions  is  emulsified 
in  a  solution  of  glycerin  and  serves  as  seed  for  inoculating  calves 
for  the  regular  supply  of  vaccine. 

Young  female  calves  from  two  to  four  months  old  that  are  certi- 
fied to  be  free  from  disease  are  prepared  for  vaccination.  The 
posterior  abdomen  and  inner  surface  of  the  thighs  are  shaved, 
washed  with  soap  and  water,  then  with  sterile  water  and  alcohol, 
and  dried  with  a  sterile  towel.  Over  this  area  a  number  of  long 
incisions  about  a  quarter  of  an  inch  apart  are  made  into  which  the 
seed  virus  is  rubbed. 

After  vaccination  the  calves  are  kept  in  specially  constructed 
stables  with  concrete  floors  and  walls;  they  stand  upon  raised 
racks  of  galvanized  iron  and  are  fed  upon  milk. 

On  the  fifth  day  the  inoculated  area  is  washed  with  sterile  water 
and  sterile  cotton  and  the  crusts  are  removed.  The  soft  pulpy 
mass  remaining  is  scraped  with  a  sterile  curette  into  a  sterilized 
container  and  mixed  with  four  times  its  weight  of  glycerin  and 
water  (glycerin  50  per  cent,  water  49  per  cent,  carbolic  acid  I  per 
cent).  The  diluted  pulp  is  passed  through  a  fine  meshed  sieve, 
after  which  it  is  tested  for  purity  by  (1)  plating  on  agar  each  week 
for  five  weeks  and  counting  the  colonies  (usually  by  the  end  of 
three  weeks  no  growth  occurs,  the  glycerin  and  carbolic  acid  hav- 
ing killed  off  all  the  contaminating  organisms) ;  (2)  animal  inoc- 
ulations to  test  for  streptococci  and  tetanus  bacilli. 

After  the  product  has  been  found  to  be  free  from  all  extraneous 
organisms  its  efficiency  is  tested  by  inoculating  fifteen  previously 
unvaccinated  children,  all  of  which  must  show  a  perfect  "  take  " 
in  order  that  the  vaccine  may  be  passed  as  up  to  standard.  After 
it  has  been  issued  for  general  use  a  clinical  test  is  made  every  two 


DISEASES  OF  UNKNOWN  ETIOLOGY  311 

weeks  during  the  period  for  which  its  potency  is  guaranteed,  and 
if  one  of  these  tests  fail  the  vaccine  is  called  in. 

The  immunity  produced  by  successful  vaccination  is  of  rela- 
tively long  duration, — it  may  last  from  ten  to  fifteen  years.  Never- 
theless it  is  well,  when  liable  to  exposure,  to  revaccinate  at  the  end 
of  a  year. 

DISEASES  OF  UNKNOWN  ETIOLOGY 

Measles.  —  Cell  inclusions,  bacilli,  and  cocci  have  all  been  de- 
scribed by  different  investigators  as  possibly  associated  with  the 
etiology  of  measles .  The  reports,  however,  have  not  been  confirmed 
and  as  yet  the  causal  agent  of  the  disease  is  unknown.  Hektoen 
in  1905  succeeded  in  experimentally  producing  measles  in  two  medi- 
cal students  by  the  subcutaneous  injection  of  blood  taken  from  a 
measles  patient  during  an  early  stage  of  the  disease.  Anderson 
and  Goldberger  in  1911  reported  having  produced  the  disease  in 
monkeys  by  means  of  a  filtrate  of  measles  blood.  All  efforts  to 
cultivate  the  virus  have  failed. 

Scarlet  Fever.  —  The  causal  agent  of  the  disease  is  still  unknown. 
Streptococci  have  been  repeatedly  found  in  large  numbers  in  the 
throats  of  scarlet  fever  patients  and  for  this  reason  have  been 
considered  by  certain  authorities  as  the  possible  inciters  of  the 
disease.  Other  workers  regard  them  merely  as  secondary  invaders. 

Mumps.  —  Although  an  infectious  disease  mumps  has  been 
little  studied ;  serum  taken  from  recovered  cases  has  been  shown 
to  contain  protective  bodies.  The  organism  giving  rise  to  the 
disease,  however,  has  not  yet  been  demonstrated. 

Rocky  Mountain  Spotted  Fever.  —  The  disease  is  characterized 
by  fever  and  an  hemorrhagic  eruption.  Diplococcoid  bodies  have 
been  described  as  present  in  the  blood  of  infected  patients.  Similar 
bodies  have  also  been  found  in  the  glands  of  ticks  who  have  fed 
upon  such  patients.  Numbers  of  these  supposed  organisms  may 
also  be  found  in  the  larvae  of  infected  female  ticks;  but  their 
causal  relationship  to  the  disease  is  not  proved. 

Chickenpox.  —  No  specific  organism  has  been  demonstrated  in 
connection  with  the  disease.  It  has  been  claimed  that  a  degree 
of  immunity  may  be  conferred  by  vaccination  with  the  clear  con- 
tents of  the  vesicles. 


INDEX 


Abscesses,      oacterial     examination     of   Animal  inoculation,  61 


material  from,  105 
Achorion  schoenleinii,  274 
Acid,  test  for  production  of,  60 
Acid-fast  bacteria,  47,  188,  189,  198 
Acids  as  disinfectants,  21 
Actinomyces,  267,  268 

cultivation  of,  269 

mode  of  infection  by,  270 

pathogenesis  of,  270 

resistance  of,  270 
Actinomycosis,  268 
^Edes  calopus  mosquito,  303 
Aerobes,  15 

facultative,  15 

obligatory,  15 
African  horse  sickness,  307 
Agar  plates,  growth  on,  59 
Agar  slant,  growth  on,  59 
Agglutination  reaction,  macroscopic,  137 

microscopic,  137 

towards  meningococcus,  171 
Agglutinins,  126,  127,  136 
Aggressins,  113 
Alcohol,  as  disinfectant,  19 
Aleppo  boil,  288 
Alexin,  128 

Allergic  skin  reactions,  151 
Allergy,  150 
Ameba,  279 
Ameba  coli,  282 
Ameba  gingivalis,  282 
Ameba  histolytica,  280 

cultivation  of,  281  -, 

examination  of  feces  for,  281 
Amebic  dysentery,  217,  280   . 
Amphitricha,  10 
Anaerobes,  15 

cultivation  of,  58 

facultative,  15 

obligatory,  15 

pathogenic,  242 

wound,  differentiation  of,  248 
Anaphylactic  shock,  151 

skin  reaction,  152 
Anaphylaxis,  150 

serum,  151 


cutaneous,  62 

intracutaneous,  62 

intraperitoneal,  62 

intravenous,  62 

methods  of,  62 

miscellaneous,  63 

subcutaneous,  62 
Animals  and  plants,  interdependence  of, 

65 
Anopheles  mosquito,  291 

differentiated  from  culex,  293,  295 
Anthrax,  221 

bacillus  of ,  22 1 ,  and  see  Bacillus  anthracis 
in  soil,  69 

intestinal,  224 
Antibiosis,  17 
Antibodies,  123,  124 

relation  to  antigens,  142 

three  orders  of,  126,  128 
Antiformin,  as  disinfectant,  21 
Antigens,  123 

relation  to  antibodies,  142 
Antiseptic  action,  17 
Antitoxin,  126 
Antitoxins,  114,  126 

production  of,  for  therapeutic  purposes, 
117 

standardization  of,  118 

unit  of,  118,  121 
Arnold  steam  sterilizer,  28 
Ascomycetes,  272 
Ascus,  272 
Aspergillus,  273 
Atricha,  10 
Attenuation,  17 
Autoclave,  26 
Autogenous  infection,  95 

vaccines,  148 
Autolysis,  112 
Autopsy  on  animals,  63 
Azobacter,  68 

Babesia,  279,  289 
Bacillary  dysentery,  217 
Bacilli,  4 

capsulated,  205 


313 


314 


INDEX 


Bacillus  aerogenes  capsulatus,  246 

in  soil,  70 
Bacillus  anthracis,  221 

cultivation  of,  222 

immunity  to,  224 

morphology  of,  222 

pathogenesis  of,  223 

resistance  of,  223 

staining  of,  222 

vaccines,  224 
Bacillus  avisepticus,  236 
Bacillus  botulinus,  249 

cultivation  of,  250 

morphology  of,  250 

pathogenesis  of,  250 

staining  of,  250 
Bacillus    of    chancroid,    235,    and    see 

Bacillus  of  soft  chancre 
Bacillus  chauvei,  248,  249 
Bacillus  coli  communis,  202 

cultivation  of,  203 

immunity  to,  204 

morphology  of,  202 

pathogenesis  of,  204 

resistance  of,  203 

staining  of,  202 

vaccines,  205 

Bacillus  coli  communior,  205 
Bacillus  diphtheria?,  177,  and  see  Diph- 
theria bacillus 
Bacillus  dysenteriae,  see  Dysentery  group 

of  bacteria 

Bacillus  edematis  maligni,  247 
Bacillus  enteritidis,  206 

sporogenes,  246 
Bacillus  fusiformis,  250 
Bacillus  Hofmanni,  186 
Bacillus  influenzas,  232 

cultivation  of,  232 

immunity  to,  234 

morphology  of,  232 

pathogenesis  of,  233 

resistance  of,  233 

staining  of,  232 

Bacillus  of  Johne's  disease,  199 
Bacillus  lactis  aerogenes,  205 
Bacillus  of  leprosy,  198 
Bacillus  of  malignant  edema,  247,  249 

cultivation  of,  248 

morphology  of,  247 

pathogenesis  of,  248 

staining  of,  247 
Bacillus  mallei,  225 

cultivation  of,  226 

diagnosis  of,  227 

mallein  reaction  in,  228 

morphology  of,  226 

pathogenesis  of,  226 

resistance  of,  226 


Bacillus  mallei  —  Continued 

serum  reaction  in,  228 

staining  of,  226 

Straus  reaction  in,  228 
Bacillus  ozense,  205 
Bacillus  perfringens,  246 
Bacillus  pertussis,  234 

cultivation  of,  235 

morphology  of,  234 

pathogenesis  of,  235 

staining  of,  234 
Bacillus  pestis,  237 

cultivation  of,  238 

immunity  to,  241 

modes  of  infection  by,  240 

morphology  of,  237 

pathogenesis  of,  238 

resistance  of,  238 

staining  of,  237 

Bacillus  phlegmonis  emphysematosis,  246 
Bacillus  pneumonise,  205 
Bacillus  proteus,  230 

cultivation  of,  230 

morphology  of,  230 

pathogenesis  of,  230 

staining  of,  230 

Bacillus  of  pseudodiphtheria,  186 
Bacillus  psittacosis,  207 
Bacillus  pyocyaneus,  229 

cultivation  of,  229 

morphology  of,  229 

pathogenesis  of,  230 

staining  of,  229 
Bacillus  rhinoscleroma,  205 
Bacillus  of  Shiga,  217 
Bacillus  of  soft  chancre,  235 

cultivation  of,  235 

morphology  of,  235 

pathogenesis  of,  236 

staining  of,  235 
Bacillus  subtilis,  225 
Bacillus  suipestifer,  207 
Bacillus  suisepticus,  236 
Bacillus  tetani,  242,  249 

antitoxin,  246 

cultivation  of,  243 

morphology  of,  242 

pathogenesis  of,  244 

resistance  of,  243 

staining  of,  242 

Bacillus  of  Timothy  grass,  199 
Bacillus  typhosus,  208 

bacterial  diagnosis  of,  215 

carriers,  212 

cultivation  of,  209 

immunity  to,  215 

modes  of  communication,  213 

morphology  of,  208 

pathogenesis  of,  210 


INDEX 


315 


Bacillus  typhosus  —  Continued 
resistance  of,  210 
serum  diagnosis  of,  215 
staining  of,  208 
vaccines,  216 
Bacillus  Welchii,  246,  249 
cultivation  of,  247 
morphology  of,  247 
pathogenesis  of,  246 
staining  of,  247 
Bacillus  xerosis,  187 
Bacteremia,  101 
Bacteria,  5 

ability  of,  to  produce  disease,  94 
acid  fast,  47,  188,  189,  198 
capsulated,  9 

chemical  composition  of,  12 
classification  of,  1,  4,  5 
composition  of,  1 
cultural  reactions  of,  59 
degenerate  forms  of,  7 
cold,  effects  of  on,  16 
cultivation  of,  51,  58 
cultural  reactions  of,  59 
defensive  forces  of  body  against,  96 
effect  of  chemicals  on,  17 
electricity  on,  15 
heat  on,  16 
light  on,  15 
food  requirements  of,  13 

test  for,  60 
general  forms  of,  5 
Gram-negative,  50 
Gram-positive,  50 
growth  of,  7 

factors  checking,  7 
factors  influencing,  13 
results  of,  22 
habitat  of,  13 
higher,  5 

identification  of,  51,  59 
in  industries,  64,  70 
in  maceration  industries,  73 
in  milk,  83,  88,  and  see  Milk 
in  natural  processes,  64 
in  suspension,  to  determine  number  of, 

147 

influence  of  body  tissues  on,  96 
involution  forms  of,  7 
lower,  5 

microscopic  examination  of,  38,  41 
morphologic  relations  of,  4 
morphology  of,  59 

determination  of,  59 
motility  of,  9 

determination  of,  59 
mutations,  8 
nitrifying,  67 
inon-pathogenic,  100 


Bacteria  —  Continued 

number  invading  body,  98 

in  cultures,  estimation  of,  55 
oxygen  requirements  of,  14 

test  for,  60 
parasites,  100 
pathogenic,  100 
pathogenic  effects  of,  100 
points  of  entrance  to  body,  97 
proteolytic  action  of,  60 
purification  of  sewage  by,  82 
reaction  of,  to  Gram's  stain,  50 
reproduction  of,  6 
saprophytes,  13,  100 
size  of,  6 
spore  formation  of,  59 

test  for,  59 
staining  of,  43 
capsules,  47 
decolorizing  agents,  45 
flagella,  48 

formulae  of  stains,  46,  and  see  Stains 
mordants,  45 
principles,  43 
saturated  solutions,  44 
spores,  47 
staining    reactions    of,    determination 

of,  59 

structure  of,  1,  8 
temperature  requirements  of,  15 
terminology  of,  5 
transition  forms,  5 
virulence  of,  99 
Bacterial  activity,  forms  of,  22 

toxins,  112 

Bacteriological  examinations,  105 
Bacteriolysins,  128,  140 
Balantidium,  279 
Balantidium  coli,  298 
Bichloride  of  mercury,   as  disinfectant, 

19 

Black  death,  237 
Blackwater  fever,  298 
Blastomycetes,  4,  275 
Blastomycosis,  276 
Bleaching  powder,  as  disinfectant,  20 

in  purification  of  water,  81 
Blepharoplast,  277 
Blood,  cultures  from,  108 
Blood  films,  43 
Blood  smear  to  make,  43 
Body,  defensive  forces  of,  96 
Boiling,  sterilization  by,  26 
Botulism,    differentiated   from   ptomain 

poisoning,  115 
Brill's  disease,  252 
Broth,  growth  on,  59 
Brownian  movement,  9 
Bubonic  plague,  237,  239 


316 


INDEX 


Budding  fungi,  275 
Butter,  bacteriology  of,  92 

Calcium    compounds,    as    disinfectants, 

20 

Calmette's  ophthalmic  test,  196 
Canning  of  food,  71 
Capsulated  bacilli,  205 

bacteria,  9 

Capsules,  staining  of,  47 
Carbolic  acid,  as  disinfectant,  18 
Carriers,  95,  104 

cholera,  256 

meningitis,  170 

pneumonia,  166 

typhoid,  212 
Cattle  plague,  307 
Caustic  soda,  as  disinfectant,  20 
Centrosome,  277 
Chagas  disease,  286 
Chancre,  soft,  235 
Chancroid,  235 

bacillus  of,  235,  and  see  Bacillus  of 

soft  chancre 

Chauveau's  theory  of  immunity,  123 
Cheese,  bacteriology  of,  93 
Chemical  effects  of  bacterial  growth,  22 
Chemicals,  effect  of,  on  bacteria,  17 
Chemotaxis,  130 
Chicken  sarcoma,  307 
Chickenpox,  311 
Chlamydospores,  272 
Chlamydozoa,  299 
Chlorid  of  lime,  as  disinfectant,  20 

in  purification  of  water,  81 
Chlorin,  as  disinfectant,  21 
Chlorinated  lime,  in  purification  of  water, 
81 

as  disinfectant,  20 
Chlorinated  soda,  as  disinfectant,  21 
Chloroform,  as  disinfectant,  19 
Cholera  carriers,  256 
Cholera  spirillum,  253 

allied  spirilla,  258 

bacteriological  diagnosis,  257 

cultivation  of,  254 

immunity  to,  257 

modes  of  transmission  of,  255 

morphology  of,  253 

pathogenesis  of,  256 

resistance  of,  255 

in  soil,  70 

staining  of,  253 

in  water,  79 
Ciliata,  4,  279,  289,  298 
Cladothrix,  267 
Cladothrix  asteroides,  267 
Claviceps  purpurea,  273 
Cocci,  4 


Coccidia,  279,  289 

Coccidium  cuniculi,  289 

Cold,  effect  of,  on  bacteria,  16 

Coley's  mixture,  161 

Colles'  law,  261 

Colon    bacilli    in    water,    determinative 

test  for,  78 

presumptive  test  for,  77 
significance  of,  75 
Colon  group  of  bacteria,  201,  202 
Colon-typhoid  group,  200,  201,  208  - 

fermentation  reactions  of,  219 
Colony  fishing,  53 
Comma  bacillus,  253,  and  see  Cholera 

spirillum 
Complement,  128 

fixation  of,  140,  141 
Conidiospores,  272 
Conjunctivitis,  234 
Conradi-Drigalsky  medium,  35 
Contagious  diseases,  95 
Contagious  pleuropneumonia  of  cattle, 

307 
Copper  sulphate,  in  purification  of  water, 

81 

Corrosive  sublimate,  as  disinfectant,  19 
Cowpox,  relation  of,  to  smallpox,  309 
Culex  mosquito,  291 

differentiated    from    anopheles,    293, 

295 

Cultural  reactions  of  bacteria,  59 
Culture,  52 

estimating  number  of  bacteria  in,  55 
method  of  inoculating,  52 
plating,  53 
pure,  52 

Culture  media,  29 
adjustment,  30 
clearing  of,  31 
filtering  of,  31 
preparation  of,  32 
agar,  33 
blood  agar,  36 
Conradi-Drigalsky,  36 
gelatin,  33 
glucose  broth,  33 
glycerin  broth,  33 
glycerin  potato,  34 
Hiss  serum  water,  37 
Loeffler's  serum,  36 
milk,  35 

neutral  red  lactose  broth,  35 
nutrient  broth,  33 
peptone  water,  34. 
potato,  34 
titration  of,  30 
tubing  of,  32 
Cytase,  128 
Cytolysins,  128,  140 


INDEX 


317 


Dark  ground  illumination,  40 
Decolorizing  agents,  45 
Delhi  sore,  288 
Deneke's  spirillum,  259 
Dengue,  307 
Denitrification,  66 
Diphtheria  antitoxin,  116 

production  of,  117 

unit  of,  121 
Diphtheria  bacillus,  177 

animal  inoculation  as  a  test  of  toxicity 
of,  185 

bacteria  resembling,  186 

bacteriological  diagnosis,  184 

cultivation  of,  179 

immunity  to,  183 

isolation  of,  179 

mixed  infection,  184 

morphology  of,  178 

pathogenesis  of,  180 

persistence  of,  in  throat,  183 

prophylactic     immunization     against, 
119 

resistance  of,  180 

staining  of,  178 

toxin  of,  115,  181 

transmitted  by  milk,  90 

virulence  of,  test  of,  185 
Diphtheroids,  187 
Diplobacilli,  7 
Diplococci,  6 
Diplococcus  intracellularis  meningitidis, 

168,  and  see  Meningococcus 
Diplococcus    pneumonias,    164,    and  see 

Pneumococcus 

Diseases,  ability  of  bacteria  to  produce, 
94 

contagious,  95 

defensive  forces  of  body  against,  96 

infectious,  95 

of  unknown  etiology,  311 
Disinfectants,  17,  18 

application  of,  21 

standardization  of,  18 
Disinfection,  17 
Dourine,  285 

Dry  heat,  sterilization  by,  25 
Drying  of  food,  71 
Dumdum  fever,  287 
Dutton,  spirochete  of,  264 
Dysentery,  216 

amebic,  217,  280 

bacillary,  217 

group  of  bacteria,  201,  208,  216 
bacteriological  diagnosis,  220 
cultivation  of,  218 
immunity  to,  219 
morphology  of,  217 
resistance  of,  218 


Dysentery  group  of  bacteria  —  Continued 
pathogenesis  of,  218 
staining  of,  217 
vaccines,  220 
types  of,  217 

Ear,  cultures  from,  108 

East  African  tick  fever,  264 

Ehrlich's  side-chain  theory  of  immunity, 

124 

Electricity,  effects  of,  on  bacteria,  15 
El  Tor  vibrios,  258 
Endospores,  10 
Endotoxins,  112 
Entameba,  278 

examination  of  feces  for,  280 
Epidemic  poliomyelitis,  301 
Epidemics,  milk-borne,  character  of,  90 
Erysipelas,  161 
Estivo-autumnal  fever,  294 
Exogenous  infection,  94 
Exotoxins,  112,  114 
Eye,  cultures  from,  108 

Farcy,  226,  227 

buds,  227 

pipes,  227 
Favus,  274 
Feces,  bacteriological  examination  of,  108 

examination  of,  for  entameba  histolyt- 

ica,  280 
Fermentation,  23 

reaction  of  colon-typhoid  group,  219 

stormy,  247 

test  for,  60 

tubes,  35 

Film  preparation,  42 
Filters,  80 

household,  81 

mechanical,  81 

sand,  80 
Filtrable  viruses,  300 

diseases  caused  by,  300 
Filtration  of  water,  80 
Finkler-Prior  spirillum,  259 
Fishing,  57 
Fission,  6 

Fixation  of  tissues,  109 
Flagella,  9 

arrangement  of,  10 

staining  of,  48 
Flagellata,  4,  278,  282 
Food  cycle  in  plants  and  animals,  67 
Food  idiosyncrasies,  152 
Food  of  bacteria,  13 
Food,  preservation  of,  role  of  bacteria 

in,  71 
Foot  and  mouth  disease,  306 

transmitted  by  milk,  89 


318 


INDEX 


Formaldehyde,  as  disinfectant,  19 
Formalin,  as  disinfectant,  19 
Frambesia,  262 
Frankel's    pneumococcus,    see   Pneumo- 

coccus 

Friedlander's  pneumobacillus,  205 
Fungi,  4 

budding,  275 

imperfecti,  273,  274 

thread,  275 

Gametocytes,  292 

Gametophores,  272 

Gas  production,  test  for,  60 

Germ,  5 

Glanders,  225,  226 

Glassware,  cleaning  of,  24 

sterilization  of,  24 
Glossina  palpalis,  286 

morsitans,  286 
Gonococcus,  171 

compared  with  meningococcus,  173 

cultivation  of,  172 

immunity  to,  174 

micrococci  resembling,  175 

morphology  of,  172 

pathogenesis  of,  173 

resistance  of,  173 

staining  of,  172 

vaccines,  175 

Gonorrhea,  Wassermann  reaction  for,  141 
Gram-negative  bacteria,  50 
Gram-positive  bacteria,  50 
Gram's  stain,  49 

reaction  of  bacteria  to,  50 
Guarnieri,  inclusion  bodies  of,  309 

Haffkine's  prophylactic,  241 

Hanging  block,  42 

Hanging-drop  preparation,  41 

Haptophore,  126 

Hay  bacillus,  225 

Heat,  effect  of,  on  bacteria,  16 

result  of  bacterial  growth,  22 
Hemameba,  290 
Hemoglobinophilic  group,  232 
Hemoglobinuria,  290 
Hemolysins,  140 
Hemolysis,  128 

Hemorrhagic  septicemia  group,  232,  236 
Hiss'  method  of  staining  capsules,  47 

serum  water,  37 
Hog  cholera,  307 
Hot-air  chamber,  25 

sterilizer,  25 
Hydrophobia,  303 
Hypersusceptibility,  150  j 
Hypha,  271 
Hyphomycetes,  4,  271 


Identification  of  bacteria,  59 
Immunity,  122 

acquired,  144 

active  acquired,  144 

cellular  theory,  124,  129 

Chauveau's  theory,  123 

Ehrlich's  side-chain  theory,  124 

following  infection,  145 

humoral  theory,  124 

mechanism  of,  124 

Metchnikoff's  theory,  124,  129 

natural,  143 

passive  acquired,  149 

Pasteur's  theory,  122 

theories  of,  122 

types  of,  143 
Immunization,  by  attack  of  disease,  145 

by  introduction  of  dead  causal  agent, 
147 

by    introduction    of    modified    causal 
agent,  145 

by  vaccines,  147 

with  toxins,  148 

Inclusion  bodies  of  Guarnieri,  309 
Incubation,  57 

period  of,  101 
Indian  ink  method  for  examination  of 

spirochetes,  48 
Indicators,  35 
Infantile  diarrhea,  transmitted  by  milk, 

89 
Infection,  94 

acute,  103 

autogenous,  95 

chronic,  103 

degrees  of,  102 

exogenous,  95 

immunity  following,  145 

malignant,  102 

mixed,  99 

secondary,  99 

spread  of,  103 

stages  of,  101 
Infectious  diseases,  95 
Infectious  jaundice,  263 
Influenza,  bacillus  of,  232,  and  see  Bacil- 
lus influenzae 

Inoculating,  method  of,  52 
Inspissation,  29 
Intestinal  anthrax,  224 
Intestinal  bacteria,  200 
lodin,  as  disinfectant,  21 
lodoform,  as  disinfectant,  19 

Jaundice,  infectious,  263 
Johne's  disease,  bacillus  of,  199 

Kala-azar,  287 
Karyosome,  277 


INDEX 


319 


Kinetic  nucleus,  277 
Koch,  spirochete  of,  264 
Koch's  postulates,  104 
Koch- Weeks  bacillus,  234 

L+,  118 

Labarraque's  solution,  as  disinfectant,  21 
Laboratory  rules,  51 
Leishman-Donovan  bodies,  287 
Leishmania,  278 

infantum,  288 

tropica,  288 

Leishmania-Donovani,  286 
Leprosy,  bacillus  of,  198 

rat,  199 

Leptothrix,  267 
Leptothrix  buccalis,  267 
Leukocidin,  156 

Leukocytes,  protective  action  of,  129, 130 
Light,  effect  of,  on  bacteria,  15 

result  of  bacterial  growth,  22 
Lime,  chlorid  of,  as  disinfectant,  20 

milk  of,  as  disinfectant,  20 
Limes  death,  118 
Litmus  milk,  35 
Loeffler's  methylene  blue  stain,  46 

serum,  36 
Lophotricha,  10 
Luetin,  262 
Lumpy  jaw,  270 
Lysins,  139 
Lysol,  as  disinfectant,  19 

Macrocytase,  129 
Macrogametocytes,  292 
Macrophages,  129 
Madura  Foot,  271 
Malaria,  290 

Malarial  parasites,  varieties  of,  294 
Malignant  edema,  bacillus  of,  247,  and 
see   Bacillus  of   malignant  edema 

in  soil,  70 

Malignant  fever,  294 
Malignant  pustule,  224 
Mallein  reaction,  228 
Mantoux's  intracutaneous  test,  196 
Massaval's  spirillum,  259 
Measles,  311 
Meat  poisoning,  249 
Media,  see  Culture  media 
Meningococcus,  168 

agglutinins,  171 

compared  with  gonococcus,  173 

cultivation  of,  169 

micrococci  resembling,  175 

morphology  of,  169 

pathogenesis  of,  170 

resistance  of,  169 

staining  of,  169 

vaccines,  171 


Merozoites,  278,  291 
Metachromatic  granules,  9 
Metazoa,  277 
Metchnikoff's  spirillum,  258 

theory  of  immunity,  129 
Microbe,  5 
Micrococci,  6 

Micrococcus  catarrhalis,  175 
Micrococcus    lanceolatus,    see    Pneumo- 

coccus 

Micrococcus  Melitensis,  175 
Micrococcus  tetragenus,  157 

cultivation  of,  157 

morphology  of,  157 

pathogenesis  of,  157 

staining  of,  157 
Microcytase,  129 
Microgametocytes,  292 
Microorganisms,  5 

acid-fast,  47,  188,  189,  198 
Microphages,  129 
Microscope,  38,  39 

double,  41 

Microsporon  furfur,  274 
Milk,  83 

bacteria  in,  83,  88 

bacteriology  of,  83 

colored,  87 

diseases  transmitted  by,  88,  90 

estimation  of  bacterial  content  in,  85 

germicidal  property  of,  84 

litmus,  35 

number  of  bacteria  in,  84,  85 

pasteurization  of,  90 

pathogenic  organisms  in,  88 

putrid,  87 

ropy,  87 

sour,  86 

standards,  86 

sterilization  of,  90 

Milk-borne  epidemics,  character  of,  90 
Milk  of  lime,  as  disinfectant,  20 
Mineralization,  66 
Minimum  lethal  dose,  117 
Mixed  infection,  153 
Moeller's  method  of  staining  spores,  47 
Moist  heat,  sterilization  by,  26 
Moisture  needed  by  bacteria,  14 
Molds,  4,  266,  271 
Monilia  Candida,  275 
Monotricha,  10 
Morax-Axenfeld  bacillus,  234 
Mordants,  45 

Moro's  percutaneous  test,  196 
Morphology  of  bacteria,  determination 

of,  59 

Mosaic  disease  of  tobacco,  307 
Mosquitoes, 

sedes  calopus,  303 


320 


INDEX 


Mosquitoes  —  Continued 

anopheles,  291,  293,  295 

culex,  291,  293,  295 

stegomyia,  303 

Motility  of  bacteria,  test  for,  59 
Mucor,  273 
Mucor  mucedo,  272 

Mucous  membranes,  bacteriological  ex- 
amination of  material  from,   105 
Mumps,  311 
Mutations,  8 
Mycetoma,  271 
Mycomycetes,  4,  271,  272 

Nagana,  285 
Negri  bodies,  304 
Neisser's  stain,  46 
Neosporidia,  4 
Nitrifying  bacteria,  67 
Nitrobacter,  67 
Nitrogen  cyc.le,  65 
Nitrosobacteria,  67 
Nitrosomonas,  67 
Nocardia,  267 
Non-malarial  cachexia,  287 
Nose,  cultures  from,  106 
Nosema,  279,  289 
Nucleus,  kinetic,  277 

Obermeier,  spirochete  of,  263 
Oidia,  4 

Oidium  albicans,  275 
Ookinete,  293 
Opsonic  index,  134 
Opsonins,  133 
Optimum  temperature,  15 
Osmosis,  14 
Osteosarcoma,  268 
O.  T.,  195 

Oxygen  requirements  of  bacteria,  14 
test  for,  60 

Parasites,  14,  100 

facultative,  14 

strict,  14 

Paratubercular  dysentery  of  cattle,  199 
Paratyphoid  bacilli,  207 

A.,  207 

B.,  207 
Paratyphoid  group  of  bacteria,  201,  205 

members  found  in  animal  diseases,  207 
Pasteur's  theory  of  immunity,  122 

treatment  of  rabies,  306 
Pasteurization  of  milk,  90 
Pathogenic  effects  of  bacteria,  100 
Pathogenic  streptococci,  158 
Pathogenic  trichomycetes,  266 
Pebrine,  289 
Penicillium,  273 
Peritricha,  10 


Pernicious  malarial  fever,  294 
Pestis  major,  240 

minor,  240 
Petri  dish,  53 

Pfeiffer's  phenomenon,  125,  128,  139 
Phagocytes,  129 
Phagocytosis,  130 
Phycomycetes,  4,  271,  272 
Phy  to  toxins,  115 
Pigment,  productive,  60 

result  of  bacterial  growth,  22 
Pink  eye,  234 
Piroplasma,  289 
Pityriasis  versicolor,  274 
Plague,  types  of,  239,  and  see  Bacillus 

pestis 
Plants  and  animals,  interdependence  of, 

65 
Plasmodia,   289,  290,  279 

asexual  phase  of,  291 

reproduction  of,  291 

schizogony,  291 

sexual  phase  of,  292 

sporogony,  292 

Plasmodium  falciparum,  294,  295 
Plasmodium  malarias,  290,  294 

cultivation  of,  296 

immunity  to,  297 

methods  of  examination,  296 

pathogenesis  of,  296 

prophylaxis  of,  297 
Plasmodium  vivax,  294,  295 
Plasmolysis,  14 
Plasmoptysis,  14 
Plating,  53,  55 
Pleuropneumonia  of  cattle,   contagious, 

307 
Pneumococci,  relation  of,  to  streptococci, 

168 
Pneumococcus,  164 

cultivation  of,  165 

immunity  to,  167 

morphology  of,  165 

pathogenesis  of,  166 

resistance  of,  166 

staining  of,  165 
Pneumococcus  mucosus,  168 
Pneumonia  carriers,  166 
Pneumonia,    microorganisms    found    in, 

164 

Pneumonic  plague,  239 
Poliomyelitis,  epidemic,  301 
Postulates,  Koch's,  104 
Potassium  permanganate,  as  disinfectant, 

20 

Potato  tube,  34 
Pour  plates,  53 
Precipitin,  128 
Precipitins,  126,  139 


INDEX 


321 


Preservation  of  food,  71 

Prof  eta's  law,  261 

Proteolytic  action  of  bacteria,  60 

Protozoa,  4,  277 

classification  of,  4 

morphology  of,  277 

nutrition  of,  278 

pathogenic,  277,  278 

reproduction  of,  278 
Pseudodiphtheria  bacilli,  186 
Pseudotuberculosis,  267 
Ptomain  poisoning,  206 

differentiated  from  botulism,  115 

from  toxins,  115 
Pure  culture,  52 

Purification  of  water,  methods  of,  80 
Pus,  bacteriological  examination  of,  106 
Putrefaction,  23 
Pyemia,  101 
Pyocyanase,  229 
Pyogenic  cocci,  153 
Pyorrhea  alveolaris,  282 

Quartan  fever,  294 

Rabies,  303 

fixed  virus,  305 

Pasteur  treatment  of,  306 

prevention  of,  304 
Radium,  effect  of,  on  bacteria,  15 
Rat  leprosy,  199 
Rat  virus,  207 
Ray  fungus,  268 

Receptors,  125,  and  see  Antibodies 
Red-water  fever,  290 
Relapsing  fever,  263 
Resistance,  122,  and  see  Immunity 
Rhizopodia,  4,  278 
Rinderpest,  307 
Ringworm,  273 

Rocky  Mountain  spotted  fever,  311 
Roentgen  rays,  effect  of,  on  bacteria,  15 
Rubber  stoppers  and  tubing,  to  cleanse,  29 

Saccharomyces  busse,  276 
Saccharomycetes,  4 
Saprophytes,  13,  100 

facultative,  14 

strict,  13 
Sarcinse,  6 

Sarcosporidia,  279,  289 
Sarcosporidiosis,  289 
Saturated  solutions  of  stains,  44 
Scarlet  fever,  311 

transmitted  by  milk,  90 
Schick  test,  119,  152 
Schizogony,  278,  291 
Schizomycetes,  4 
Sensitization,  128,  141 


Septic  sore  throat,  89 
Septicemia,  101 
Septicemic  plague,  239 
Serum,  method  of  obtaining,  37 
Serum  anaphylaxis,  151 
Serum  sickness,  151 

Sewage,  bacteriological  examination  of, 
74,82 

purification  of,  by  bacteria,  82 

streptococci  of,  in  water,  78 
Shiga's  bacillus,  217 
Slaked  lime,  as  disinfectant,  20 
Sleeping  sickness,  285 
Smallpox,  307 

relation  of,  to  cowpox,  309 

vaccination  against,  308 
Smegma  bacillus,  199 
Soda,  caustic,  as  disinfectant,  20 

chlorinated,  as  disinfectant,  21 

washing,  as  disinfectant,  20 
Sodium  bicarbonate  as  disinfectant,  20 

carbonate  as  disinfectant,  20 

hydroxide  as  disinfectant,  20 
Soil,  examination  of,  69 

pathogenic  bacteria  in,  69 
Solid  organs,  bacteriological  examination 

of,  109 
Spirilla,  4,  6 
Spirillum,  5 

cholera,  in  soil,  70 

Deneke,  259 

Finkler-Prior,  259 

Massaval,  259 

Metchnikovii,  258 
Spirocheta  duttoni,  264 
Spirocheta  icterohemorrhagica,  262 
Spirocheta  kochi,  264 
Spirocheta  obermeieri,  263 

cultivation  of,  263 

immunity  to,  264 

morphology  of,  263 

pathogenesis  of,  263 

staining  of,  263 
Spirocheta  pallida,  259 
Spirocheta  recurrentis,    263 
Spirochetes,  6,  259 

India  ink  method  for  examination  of, 
48 

varieties  of,  264 
Splenic  fever,  221 
Spore  formation,  10 

significance  of,  11 

tests  for,  12,  59 
Spores,  staining  of,  47 
Sporoblast,  293 
Sporocyst,  293 
Sporogeny,  278,  292 
Sporotrichosis,  274 
Sporozoa,  278,  289 


322 


INDEX 


Sporozoites,  293 

Sputum,  bacteriological  examination  of, 

106 
Stab  culture,  53 

growth  in,  59 
Staining,  of  flagella,  48 

of  spores,  47 

principles  of,  43 
Stains,  formulae  of,  46 

Gram's,  49,  and  see  Gram's  stain 

Loeffler's  methylene  blue,  46 

Neisser's,  46 

Wright's,  48 

Ziehl-Neelsen's  carbol  fuchsin,  46 

saturated  solutions  of,  44 
Staphylococci,  6 
Staphylococcus  aureus,  154 

cultivation  of,  154 

immunity  to,  156 

morphology  of,  154 

pathogenesis  of,  155 

resistance  of,  155 

staining  of,  154 

Staphylococcus  epidermidis  albus,  157 
Staphylococcus  pyogenes  albus,  156 
Staphylococcus    pyogenes    aureus,    154, 

and  see  Staphylococcus  aureus 
Staphylococcus  pyogenes  citreus,  157 
Staphylolysin,  156 

Steam  at  high  pressure,  sterilization  by,  26 
Stegomyia  mosquito,  303 
Sterilization,  17 

by  dry  heat,  25 

by  moist  heat,  26 

by  steam  at  high  pressure,  26 

discontinuous,  27 

fractional,  27 

intermittent,  27 

of  glassware,  24 

of  milk,  90 

Storage,  purification  of  water  by,  80 
Stormy  fermentation,  247 
Straus  reaction,  228 
Streptobacilli,  7 
Streptococci,  6 

pathogenic,  158 

relation  of,  to  pneumococci,  168 
Streptococcus  mucosus,  168 
Streptococcus  pyogenes,  158 

cultivation  of,  159 

immunity  to,  162 

morphology  of,  158 

pathogenesis  of,  160 

resistance  of)  160 

staining  of,  -158 

vaccines,  163 
Streptothrix,  266 
Subculture,  52 
Sugars,  fermentation  of,  60 


Sulphur  dioxid,  as  disinfectant,  21 

Surface  streaking,  54 

Surra,  285 

Susceptibility,  122 

Symbiosis,  17 

Syphilis,  259 

Wassermann  reaction  for,  142,  262 

T-A..  119 

Tabardillo,  252 

Taxis,  10 

Telosporidia,  4 

Temperature,  effect  of,  on  bacteria,  15 

optimum,  15 
Tertian  fever,  294 
Tetanolysin,  120 
Tetanospasmin,  120 
Tetanus  antitoxin,  production  of,    121 

unit  of,  121 

Tetanus,  bacillus  of,  242,  249,  and  see 
Bacillus  tetani 

in  soil,  69 

toxin,  120 
Tetrads,  6 
Texas  fever,  290 
Thread  fungi,  275 
Throat,  culture  from,  106 
Thrush,  275 

fungus,  275 
Tick  fever,  290 

East  African,  264 
Timothy  grass  bacillus,  199 
Tinea  circinata,  273 
Tinea  tonsurans,  273 
Tissues,  cutting  of,  110 

embedding  of,  110 

examination  of  bacteria  in,  109 

fixation  of,  109,  110 

hardening  of,  110 
Tobacco,  mosaic  disease  of,  307 
Toxins,  bacterial,  112 

exogenous,  114 

immunization  with,  148 

true,  114 

differentiated  from  ptomain  poison, 

115 

Toxin  unit,  117 
Toxophore,  126 
Transplant,  52 
Trench  fever,  307 
Treponema  pallidum,  259 

cultivation  of.  260 

immunity  to,  261 

luetin  reaction,  262 

microscopic  examination  of,  261 

morphology  of,  259 

pathogenesis  of  260 

staining  of,  259 

Wassermann  reaction,  262 


INDEX 


323 


Treponema  pertenue,  262 
Trichobacteria,  4,  5,  266 
Trichophyta,  273 
Tropical  ulcer,  288 
Trypanosoma,  278 
Trypanosome  brucei,  285 

equiperdum,  285 

evansi,  285 

gambiense,  285 

lewisi,  284  '} 

rhodesiense,  286 
Trypanosomes,  283 
Trypanosomiasis,  283,  285 
Tsetse  fly  disease,  285 
Tubercle  bacilli  in  milk,  88,  89 
Tubercle  bacillus,  188 

avian,  197 

bovine,  197 

cultivation  of,  189 

fish,  197 

heredity,  193 

human,  197 

immunity  to,  194 

modes  of  infection  by,  191 

morphology  of,  188 

pathogenesis  of,  190 

resistance  of,  190 

staining  of,  188 

tuberculin  in,  195,  and  see  Tuberculin 

varieties  of,  197 
Tuberculin,  as  a  diagnostic  agent,  195 

cutaneous  test  of  von  Pirquet,  196 

dose  of,  195 

intracutaneous  test  of  Mantoux,   196 

ophthalmic  test  of  Calmette,  196 

percutaneous  test  of  Moro,  196 

preparations  of,  195 

reaction,  195 

Tuberculosis  transmitted  by  milk,  88,  89 
Typhoid  bacillus,  208,  and  see  Bacillus 
typhosus 

in  milk,  90 

in  soil,  70 

in  water,  79 
Typhoid  carriers,  212 
Typhus  fever,  251 

Ulceromembranous  angina,  250 
Ultramicroscopic  organisms,  4,  300 

viruses,  3,  300 

Ultraviolet  rays  in  purification  of  water,  82 
Unit,  of  antitoxin,  118,  121 

of  toxin,  117 
Urine,  bacteriological  examination  of,  107 

Vaccination  against  smallpox,  308 
preparation  of  virus  for,  310 

Vaccine  bodies,  309 

Vaccines,  autogenous,  148 
immunization  by,  147 


Vaccines  —  Continued 

polyvalent,  148 

sensitized,  148 

Vaccinia,  relation  of,  to  variola,  309 
Van    Ermengen's    method    of    staining 

flagella,  48 
Variola,  307 

relation  to  vaccinia,  309 
Vibrios,  6 

El  Tor,  258 
Vincent's  angina,  250 
Vinegar  making,  72 
Virulence  of  bacteria,  98 
Viruses,  filtrable,  3,  300 

ultramicroscopic,  3,  300 
Von  Pirquet's  cutaneous  test,  196 

Washing  soda  as  disinfectant,  20 
Wassermann  reaction,  141,  142,  262 

in  gonorrhea,  141 

in  syphilis,  262 

Water,    bacteriological   examination   of, 
74,  76 

cholera  spirillum  in,  79 

collecting  samples  for  analysis,  77 

colon  bacilli  in,  significance  of,  75 
tests  for,  77,  78 

filtration  of,  80 

natural,  74 

purification  of,  79,  80,  81 

quantitative  analysis  of,  77 

relative  purity  of,  75 

sewage  streptococci  in,  78 

significance  of  colon  bacilli  in,  75 

storage  of,  80 

typhoid  bacilli  in,  79 
Weil's  disease,  263 
Welsh's  gas  bacillus,  in  soil,  70 
West  African  tick  fever,  264 
Whooping  cough,  234,  and  see  Bacillus 

pertussis 
Widal's  test,  136 
Wolffhugel's  counting  plate,  56 
Wool  sorters'  disease,  224 
Wounds,  bacteriological  examination  of 

material  from,  105 

Wright's  pipette  and  tube  for  opsonic 
index,  134 

stain,  48 
Yaws,  262 
Yeast  cells,  275 
Yeasts,  4,  266,  275,  276 

pathogenic,  276 
Yellow  fever,  302 

Ziehl-Neelsen's  carbol  fuchsin  stain,  46 
Zooglcea,  8 
Zootoxins,  115 
Zygote,  293 
Zymophore,  127 


Printed  in  the  United  States  of  America. 


DATE    DUE    SLIP 

UNIVERSITY   OF  CALIFORNIA   MEDICAL  SCHOOL  LIBRARY 


THIS  BOOK  IS  DUE   ON  THE   LAST   DATE 
STAMPED   BELOW 


DEC  &  - 
FEB  1  2    1930 

N8V  6     1930 
DEC   3     1931 

ylC  21  1931 

JAN  26  1932 
Feb  2  '32 
Feb  9  '32 
Fe624  '32 

SEP    12  1-331 

,.48  1938 

" 

MA'         :)38 

1  1939' 
APR  25  1933 


JUL 


1  m-7,'25 


-i6     Smeoton,   M.A. 
S6>5  Bacteriolog 


1922 
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SOS 


>t-C^V*«->  ^, 


DEC  1  0  1924 


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y  for  nur- 


AUG  2  0  I.Q9 


J12   1930 


Library  of  the 

University  of  California  Medical  School 
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